Liver enriched transcription factor

ABSTRACT

HNF-4 (hepatocyte nuclear factor 4) is a protein enriched in liver extracts that binds to sites required for the transcription of the transthyretin (TTR) and apolipoprotein CIII (apoCIII) genes (Costa et al., 1989; Costa et al., 1990; Leff et al., 1989). We have purified HNF-4 protein (54 kD) and isolated a cDNA clone encoding the protein. HNF-4 is a member of the steroid hormone receptor superfamily with an unusual amino acid in the conserved “knuckle” of the first zinc finger (DGCKG). This and the fact that HNF-4 does not bind significantly to estrogen, thyroid hormone or glucocorticoid response elements indicate that HNF-4 may represent a new subfamily. HNF-4 binds to its recognition site as a dimer and activates transcription in a sequence-specific fashion in nonhepatic (HeLa) cells. HNF-4 mRNA is present in kidney and intestine as well as liver but is absent in other tissues. DNA binding data suggest that HNF-4 could be identical to liver factor A1 (LF-A1), a factor previously shown to regulate the transcription of the α-1 antitrypsin, apolipoprotein A1 and pyruvate kinase genes.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of application U.S. Ser. No.09/447,034 filed 22 Nov. 1999, now U.S. Pat. No. 6,500,672, issued 31Dec. 2002, which is a divisional application of U.S. Ser. No. 09/038,217filed 11 Mar. 1998, now U.S. Pat. No. 6,025,196, issued 15 Feb. 2000,which is a divisional application of U.S. Ser. No. 08/661,330 filed 14Jun. 1996, now U.S. Pat. No. 5,849,485, issued 15 Dec. 1998, which is adivisional application of U.S. Ser. No. 08/078,222, filed 28 Oct. 1993,now U.S. Pat. No. 5,604,115, issued 18 Feb. 1997, which is a nationalfiling of PCT/US91/09733, filed 23 Dec. 1991, which is a PCT filing ofU.S. Ser. No. 07/631,720, filed 21 Dec. 1990, now abandoned, thedisclosures of which are herein specifically incorporated by referenceherein in their entireties. Applicants claim the benefits of theseapplications under 35 U.S.C. §§120, and 371.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to liver-related transcription factorsand, in particular, to such factors as participate in the regulation ofa variety of genes such as certain of the apolipoproteins involved infat and cholesterol transport.

This invention also relates to antibodies which recognize the receptorHNF-4, and antiidiotype antibodies that recognize both antibodies toHNF-4 and ligands which bind to HNF-4.

The invention also relates to antisense DNA and RNA moleculescomplementary to mRNA for HNF-4, and ribozymes which recognize the mRNA.

The invention also relates to methods of use of the aforementionedmolecules, DNA sequences, antibodies, anti-idiotype antibodies,antisense molecules and ribozymes, for example in developing diagnosticand therapeutic agents to detect, inhibit or enhance binding to HNF-4.

It is a principal object of this invention to provide new means tostudy, diagnose, prevent and treat disease. More particularly, it is anobject of this invention to provide molecules involved in binding toHNF-4, and to isolate other molecules which are themselves useful ininhibiting such binding.

This invention provides DNA sequences that code on expression for HNF-4,genomic DNA sequences for HNF-4, recombinant DNA molecules containingthese DNA sequences, unicellular hosts transformed with these DNAmolecules, processes for producing such receptors, and proteinsessentially free of normally associated animal proteins. The presentinvention also provides for antibody preparations reactive for HNF-4.

Monoclonal antibodies recognizing ligands to HNF-4 can inhibit ligandbinding directly or by binding or otherwise interacting with a thirdmolecule. Such molecules may act, for example, by changing the surfaceconformation of the ligand so that its affinity for the HNF-4 isreduced.

This invention also provides recombinant DNA molecules containing HNF-4DNA sequences and unicellular hosts transformed with them. It alsoprovides for HNF-4 proteins essentially free of normally associatedanimal proteins, methods for producing HNF-4, and monoclonal antibodiesthat recognize HNF-4.

This invention further provides methods for using antisense nucleicacids and ribozymes to inhibit HNF-4 expression. The invention alsorelates to methods for identifying binding inhibitors by screeningmolecules for their ability to inhibit binding of HNF-4 to its ligand.It provides methods for identifying ligands. One such method involvesusing anti-idiotypic antibodies against antibodies that recognize HNF-4or HNF-4 ligands.

BACKGROUND OF THE INVENTION

Cell type specificity is based on differential gene expression which isin turn determined, at least in part, by the particular set oftranscription factors present and active in a given cell at a giventime. Many such factors have been identified and characterized,particularly in the liver where there is a wide range oftranscriptionally controlled genes (McKnight & Palmiter, 1979; Derman etal., 1981). Some transcription factors, such as AP-1 and Sp-1, seem tobe present in all cells at all times but other factors have a morelimited distribution. Whether there is a discernible logic that explainsthe distribution of the many factors has yet to be determined. Twoaspects of this problem are particularly important. The first aspect isto determine whether the distribution of factors in different issues iscontrolled at the level of transcription. If so, then a cascade oftranscriptional regulation that ultimately results in cell specificityis indicated. The second issue is whether any particular factor iscentral to the accomplishment of a particular metabolic or physiologicgoal. Such a goal might be suggested by factors acting on aninterrelated set of genes.

These issues have begun to be addressed by the dissection and analysisof the promoter/enhancer regions of genes expressed primarily inhepatocytes by the present applicants and others (Johnson, 1990). TheDNA elements that confer cell specific expression have been defined bytransient transfection into cultured cells (e.g., hepatoma vs. HeLacells) and/or in vitro transcription assays, and the proteins that bindto these elements have been identified by DNA binding assays using crudeliver nuclear extracts. In this way, at least four distinct proteinfactors that are abundant in liver have been found thus far: HNF1(LF-B1) (Courtois et al., 1987; Monaci et al., 1988), C/EBP (Johnson etal., 1987), HNF-3 and HNF-4 (Costa et al., 1989). HNF1, a homeo domainprotein (Frain et al., 1989; Baumhueter et al., 1990), C/EBP, theoriginal leucine zipper protein (Landschulz et al., 1988), and mostrecently HNF-3A, a DNA binding protein that has no similarity to knowntranscription factor families (Lai et al., 1990) have all been purifiedand cloned so that distribution and regulation of each can bedetermined.

The following publications are cited in the body of the patentapplication. Each of the publications is incorporated herein byreference:

Ahe, von der D., Janich, S., Scheidereit, C., Renkawitz, R., Schutz, G.,and Beato, M. (1985). Glucocorticoid and progesterone receptors bind tothe same sites in two hormonally regulated promoters. Nature, 313,706-709.

Baumhueter, S., Mendel, D. B., Conley, P. B., Kuo, C. J., Turk, C.,Graves, M. K., Edwards, C. A., Courtois, G., and Crabtree, G. R. (1990).HNF-1 shares three sequence motifs with the POU domain proteins and isidentical to LF-B1 and APF. Genes and Development 4, 372-379.Beato, M. (1989). Gene regulation by steroid hormones. Cell 56, 335-344.Birkenmeier, E. H., Gwynn, B., Howard, S., Jerry, J., Gordon, J. I.,Landschulz, W. H., and McKnight, S. L. (1989). Tissue-specificexpression, developmental regulation and mapping of the gene encodingCCAAT/enhancer binding protein. Genes and Development, 3, 1146-1156.Brand, N., Petkovich, M., Krust, A., Chambon, P., de The, H., Marchio,A., Tiollais, P., and Dejean, A. (1988). Identification of a secondhuman retinoic acid receptor. Nature, 332, 850-853.Breslow, J. (1988). Apolipoprotein genetic variation and human disease.Physiol. Reviews, 68, 85-132.Capon, D. J. et al. (1989). Designing CF4 immunoadhesins for AIDStherapy. Nature, 337, 525-531.Carlsson, R., and Glad, C. (June, 1989). Monoclonal antibodies into the'90s. Bio/Technology, 7, 567-573.Cate, R. et al. (1986). Isolation of the bovine and human genes forMullerian inhibiting substance and expression of the human gene inanimal cells. Cell, 45, 685-598.Cech, T. R. (1988). Ribozymes and their medical implications. J. Amer.Med. Assn., 260, 3030-3044.Chomezynski, P. and Sacchi, N. (1987). Single-step method of RNAisolation by acid guanidinium thiocyanate-phenol-chloroform extraction.Anal. Biochem., 162, 156-159.Costa, R. H., Lai, E., and Darnell, J. E., Jr. (1986). Transcriptionalcontrol of the mouse prealbumin (transthyretin) gene: both promotersequences and a distinct enhancer are cell specific. Mol. and Cell.Biol., 6, 4697-4708.Costa, R. H., Grayson, D. R., Xanthopoulos, K. G., and Darnell, J. E.,Jr. (1988). A liver-specific DNA-binding protein recognizes multiplenucleotide sites in regulatory regions of transthyretin, a1-antitrypsin,albumin, and simian virus 40 genes. Proc. Natl. Acad. Sci., 85,3840-3844.Costa, R. H., Grayson, D. R. and Darnell, J. E. Jr. (1989). Multiplehepatocyte-enriched nuclear factors function in the regulation oftransthyretin and a1-antitrypsin genes. Mol. and Cell. Biol., 9,1415-1425.Costa, R. H., Van Dyke, T. A., Yan, C., Kuo, F., and Darnell, J. E., Jr.(1990). Similarities in transthyretin gene expression and differences intranscription factors: liver and yolk sac compared to choroid plexus.Proc. Natl. Acad. Sci. USA, 87, 6589-6593.Courtois, G., Morgau, J. G., Campbell, L. A., Fourel, G. and Crabtree,G. R. (1987). Interaction of a liver-specific nuclear factor with thefibrinogen and a1-antitrypsin promoters. Science, 238, 688-692.Danielsen, M., Hinck, L., and Ringold, G. M. (1989). Two amino acidswithin the knuckle of the first zinc finger specify DNA response elementactivation by the glucocorticoid receptor. Cell, 57, 1131-1138.Davis, M. M. (1986). Subtractive cDNA hybridization and the T-cellreceptor gene. Handbook of Experimental Immunology in Four Volumes, 4thed. Blackwell Scientific Publications, Oxford, England, 76.1-76.13.Davis, M. M. et al. (1984). Cell type-specific cDNA probes and themurine I region: The localization and orientation of ad. Proc. Natl.Acad. Sci. USA, 81, 2194-2198.Derman, E., Krauter, K., Walling, L., Weinberger, C., Ray, M. andDarnell, J. E. Jr. (1981). Transcriptional control in the production ofliver-specific mRNAs. Cell, 23, 731-739.de The, H., Marchio, A., Tiollais, P., and Dejean, A. (1987). A novelsteroid thyroid hormone receptor-related gene inappropriately expressedin human hepatocellular carcinoma. Nature, 330, 667-670.Duguid, J. R. et al. (1988). Isolation of cDNAs of scrapie-modulatedRNMAs by subtractive hybridization of a cDNA library. Proc. Natl. Acad.Sci. USA, 85, 5738-5742.Evans, R. M. (1988). The steroid and thyroid hormone receptorsuperfamily. Science, 240, 889-895.Fawell, S. E., Lees, J. A., White, R. and Parker, M. G. (1990).Characterization and colocalization of steroid binding and dimerizationactivities in the mouse estrogen receptor. Cell, 60, 953-962.Feinberg, A. P., and Vogelstein, B. (1983). A technique forradiolabeling DNA restriction endonuclease fragments to high specificactivity. Anal. Biochem., 132, 6-13.Fisher, R. A. et al. (1988). HIV infection is blocked in vitro byrecombinant soluble CD4. Nature, 331, 76-78.Forman, B. M., Yan, C. R., Au, M., Casanova, J., Ghysdael, J., andSamuels, H. H. (1989). A domain containing leucine-zipper-like motifsmediate novel in vivo interactions between the thyroid hormone andretinoic acid receptors. Mol. End., 3, 1610-1626.Forman, B. M. and Samuels, H. H. (1990). Interactions among a subfamilyof nuclear hormone receptors: The regulatory zipper model. Mol. End. 4,1293-1301.Frain, M., Swart, G., Monaci, P., Nicosia, A., Stampfli, S., Frank, R.,and Cortese, R. (1989). The liver-specific transcription factor LF-B1contains a highly diverged homeobox DNA binding domain. Cell, 59,145-157.Fried, M. and Crothers, D. M. (1981). Equilibria and kinetics of lacrepressor-operator interactions by polyacrylamide gel electrophoresis.Nucleic Acids Res., 9, 6505-6525.Giguere, V., Yang, N., Segui, P., and Evans, R. M. (1988).Identification of a new class of steroid hormone receptors. Nature, 331,91-94.Glass, C. K., Lipkin, S. M., Devary, O. V., and Rosenfeld, M. G. (1989).Positive and negative regulation of gene transcription by a retinoicacid-thyroid hormone receptor heterodimer. Cell, 59, 697-708.Gorman, C. M., Moffat, L. F. and Howard, B. H. (1982). Recombinantgenomes which express chloramphenicol acetyltransferase in mammaliancells. Mol. Cell. Biol., 2, 1044-1051.Gorman, C. M., Howard, B. H. and Reeves, R. (1983). Expression ofrecombinant plasmids in mammalian cells is enhanced by sodium butyrate.Nucleic Acids Res., 11, 7631-7648.Gorski, K., Carneiro, M. and Schibler, U. (1986). Tissue-specific invitro transcription from the mouse albumin promoter. Cell, 47, 767-776.Green, N., Alexander, H., Olson, A., Alexander, S., Shinnick, T. M.,Sutcliffe, J. G., and Lerner, R. A. (1982). Immunogenic structure of theinfluenza virus hemagglutinin. Cell, 28, 477-487.Green, S., Walter, P., Kumar, V., Krust, A., Bornert, J. M., Argos, P.,and Chambon, P. (1986). Human oestrogen receptor cDNA: Sequence,expression and homology to c/epb. Nature, 320, 134-139.Green, S. and Chambon, P. (1988). Nuclear receptors enhance ourunderstanding of transcription regulation. Trends Genet., 4, 309-314.Gubler, U., and Hoffman, B. J. (1983). A simple and very efficientmethod for generating cDNA libraries. Gene, 25, 263-269.Hamada, K., Gleason, S. L., Levi, B. Z., Hirschfeld, S., Appella, E.,and Ozato, K. (1989). H-2RIIBP, a member of the nuclear hormone receptorsuperfamily that binds to both the regulatory element of majorhistocompatibility class I genes and the estrogen response element.Proc. Natl. Acad. Sci. USA, 86, 8289-8293.Hambor, J. E. et al. (1988). Functional consequences of antisenseRNA-mediate inhibition of CDB surface expression in a human T cellclone. J. Exp. Med., 168, 1237-1245.Hardon, E. M., Frain, M., Paonessa, G. and Cortese, R. (1988). Twodistinct factors interact with the promoter regions of severalliver-specific genes. The EMBO J., 7, 1711-1719.Harlow, E. and Lane, D. (1988). Antibodies: A laboratory manual. (ColdSpring Harbor, N.Y.: Cold Spring Harbor Lab.)Hasselhoff, J., and Gerlach, W. L. (1988). Simple RNA enzymes with newand highly specific endoribonuclease activities. Nature, 334, 585-591.Hedrick, S. M. et al. (1984). Isolation of cDNA clones encoding Tcell-specific membrane-associated proteins. Nature, 308, 149-153.Ito, Y., Azrolan, N., O'Connell, A., Walsh, A., and Breslow, J. L.(1990) Hypertriglyceridemia as a result of human apoCIII gene expressionin transgenic mice. Science, 249, 790-793.Johnson, P. F., Landschulz, W. H., Graves, B. J., and McKnight, S. L.(1987). Identification of a rat liver nuclear protein that binds to theenhancer core element of three animal viruses. Genes and Development, 1,133-146.Johnson, P. F. (1990). Transcriptional activators in hepatocytes. InCell Growth and Differentiation, 1, 47-52.Kadonaga, J. T., and Tjian, R. (1986). Affinity purification ofsequence-specific DNA binding proteins. Proc. Natl. Acad. Sci. USA, 83,5889-5893.Kennedy, R. C. et al. (July, 1986). Anti-idiotypes and immunity. Sci.Am., 255, 48-56.Kozak, M. (1987). An analysis of 5′-noncoding sequences from 699vertebrate messenger RNA's. Nucleic Acids Res., 15, 8125-8143.Krebs, E., Eisenman, R., Kuenzel, E., Litchfield, D., Lozeman, F.,Lischer, B. and Sommercorn, J. (1988). Casein kinase II as a potentiallyimportant enzyme concerned with signal transduction. In MolecularBiology of Signal Transduction. (Cold Spring Harbor, N.Y.: Cold SpringHarbor Laboratory), p. 77-84.Kumar, V., and Chambon, P. (1988). The estrogen receptor binds tightlyto its responsive element as a ligand-induced homodimer. Cell, 55:145-156.Kuo, C. F., Xanthopoulos, K. G., and Darnell, J. E. Jr. (1990). Fetaland adult localization of C/EBP: evidence for combinatorial action oftranscription factors in cell-specific gene expression. Development,109, 473-481.Lai, E., Prezioso, V. R., Smith, E., Litvin, O., Costa, R. H., andDarnell, J. E., Jr. (1990). HNF-3A, a hepatocyte-enriched transcriptionfactor of novel structure is regulated transcriptionally. Genes andDevelopment, 4, 1427-1436.Landschulz, W. H., Johnson, P. F., Adashi, E. Y., Graves, B. J., andMcKnight, S. L. (1988). Isolation of a recombinant copy of the geneencoding C/EBP. Genes and Development, 2, 786-800.Lathe, E. (1985). Synthetic oligonucleotide probes deduced from aminoacid sequence data: Theoretical and practical considerations. J. Mol.Biol., 183, 1-12.Leff, T., Reue, K., Melian, A., Culver, H., and Breslow, J. L. (1989). Aregulatory element in the ApoCIII promoter that directs hepatic specifictranscription binds to proteins in expressing and nonexpressing celltypes. The J. of Biol. Chem., 264, 16132-16137.Lew, D. J., Decker, T., Strehlow, I. and Darnell, J. E. (1990).Overlapping elements in the GBP gene promoter mediate transcriptionalinduction by alpha and gamma-interferon. Mol. Cell Biol., in press.Li, Y., Shen, R. -F., Tsai, S. Y., and Woo, S. L. C. (1988). Multiplehepatic trans-acting factors are required for in vitro transcription ofthe human alpha-1-antitrypsin gene. Mol. and Cell. Biol., 8, 4362-4369.MacGregor, G. R., and Caskey, C. T. (1989). Construction of plasmidsthat express E. coli b-galactosidase in mammalian cells. Nucleic AcidsRes., 17, 2365.Mader, S., Kumar, V., de Verneuil, H., and Chambon, P. (1989). Threeamino acids of the oestrogen receptor are essential to its ability todistinguish an oestrogen from a glucocorticoid-responsive element.Nature, 338, 271-274.Mangelsdorf, D. J., Ong, E. S., Dyck, J. A. and Evans, R. M. (1990).Nuclear receptor that identifies a novel retinoic acid response pathway.Nature, 345, 224-229.Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982). Molecularcloning: A laboratory manual. (Cold Spring Harbor, N.Y.: Cold SpringHarbor Lab.)Marcus-Sekura, C. J. (1988). Techniques for using antisenseoligonucleotides to study gene-expression. Anal. Biochem., 172, 289-295.Matsudaira, P. (1987). Sequence from picomole quantities of proteinselectroblotted onto polyvinylidene difluorid membranes. The J. of Biol.Chem., 262, 10035-10038.McKnight, G. S., and Palmiter, R. D. (1979). Transcriptional regulationof the ovalbumin and conalbumin genes by steroid hormones in chickoviduct. J. Biol. Chem., 254, 9050-9058.Mermod, N., O'Neill, E. A., Kelly, T. J. and Tjian, R. (1989). Theproline-rich transcriptional activator of CTF/NF-1 is distinct from thereplication and DNA binding domain. Cell, 58, 741-753.Miyajiima, N., Kadowaki, Y., Fukushige, Shiminizu, S., Semba, K.,Yamanashi, Y. H., Matsubara, K., Toyoshima, K., and Yamanoto, T. (1988).Identification of two novel members of erbA superfamily by molecularcloning: The gene products of the two are highly related to each other.Nucleic Acids Res., 16:11057-11074.Monaci, P., Nicosia, A., and Cortese, R. (1988). Two differentliver-specific factors stimulate in vitro transcription from the humana1-antitrypsin promoter. The EMBO J., 7, 2075-2087.Mueller, C. R., Maire, P., and Schibler, U. (1990). DBP, aliver-enriched transcriptional activator, is expressed late in ontogenyand its tissue specificity is determined posttranscriptionally. Cell,61, 279-291.Pearson, W. R., and Lipman, D. J. (1988). Improved tools for biologicalsequence comparison. Proc. Natl. Acad. Sci. USA, 85, 2444-2448.Puissant, C., and Houdebine, L. M. (1990). An improvement of thesingle-step method of RNA isolation by acid guanidiniumthiocyanate-phenol-chloroform extraction. BioTechniques, 8, 148-149.Reue, K., Leff, T., and Breslow, J. L. (1988). Human apolipoprotein CIIIgene expression is regulated by positive and negative Cis-actingelements and tissue-specific protein factors. The J. of Biol. Chem.,263, 6857-6864.Rosen, C. A., Sodroski, J. G. and Haseltine, W. A. (1985). The locationof cis-acting regulatory sequences in the human T cell lymphotropicvirus type III (HTLV-III/LAV) long terminal repeat. Cell, 41, 813-823.Ruppert, S., Boshart, M., Bosch, F. X., Schmid, W., Fournier, R. E. K.,and Schutz, G. (1990). Two genetically defined trans-acting locicoordinately regulate overlapping sets of liver-specific genes. Cell,61, 895-904.Ryseck, R. P., Macdonald-Bravo, H., Mattei, M. G., Ruppert, S., andBravo, R. (1989). Structure, mapping and expression of a growth factorinducible gene encoding a putative nuclear hormonal binding receptor.The EMBO J., 8, 3327-3335.Saiki, R. K., Gelfand, D. H., Stoffel, S., Scharf, S. J., Higuchi, R.,Horn, G. T., Mullis, K. B., and Erlich, H. A. (1988). Primer-directedenzymatic amplification of DNA with a thermostable DNA polymerase.Science, 239, 487-491.Sanger, F., Nicklen, S., and Coulson, A. R. (1977). DNA sequencing withchain terminating inhibitors. Proc. Natl. Acad. Sci. USA, 74, 5463-5467.Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989). Molecularcloning: A laboratory manual. (Cold Spring Harbor, N.Y.: Cold SpringHarbor Laboratory).Sargent, T. D. (1987). Isolation of differentially expressed genes.Methods in Enzymol., 152, 423-447.Schule, R., Umesono, K., Mangelsdorf, D. J., Bolado, J., Pike, J. W.,and Evans, R. M. (1990). Jun-fos and receptors for vitamins A and Drecognize a common response element in the human osteocalcin gene. Cell,61, 497-504.Seed, B. (1987). An LFA-3 cDNA encodes a phospholipid-linked membraneprotein homologous to its receptor CD2. Nature, 329, 840-842.Seed, B., and Aruffo, A. (1987). Molecular cloning of the CD2 antigen,the T-cell erythrocyte receptor, by a rapid immunoselection procedure.Proc. Natl. Acad. Sci. USA, 84, 3365-3369.Tsai, S. Y., Carlstedt-Duke, J., Weigel, N. L., Dahlman, K., Gustafsson,J. A., Tsai, M. J., and O'Malley, B. W. (1988). Molecular interactionsof steroid hormone receptor with its enhancer element: Evidence forreceptor dimer formation. Cell, 55, 361-369.Umesono, K., Giguere, V., Glass, C. K., Rosenfeld, M. G., and Evans, R.M. (1988). Retinoic acid and thyroid hormone induce gene expressionthrough a common responsive element. Nature, 336, 262-265.Umesono, K. and Evans, R. M. (1989). Determinants of target genespecificity for steroid/thyroid hormone receptors. Cell, 57, 1139-1146.Vaulont, S., Puzenat, N., Kahn, A., and Raymondjean, M. (1989). Analysisby cell-free transcription of the liver-specific pyruvate kinase genepromoter. Mol. and Cell. Biol., 9, 4409-4415.Wang, L. H., Tsai, S. Y., Cook, R. G., Beattie, W. G., Tsai, M. J. andO'Malley, B. W. (1989). COUP transcription factor is a member of thesteroid receptor superfamily. Nature, 340, 163-166.Weinberger, C., Thompson, C. C., Ong, E. S., Lebo, R., Gruo, D. J., andEvans, R. M. (1986). The c/epb gene encodes a thyroid hormone receptor.Nature, 234, 641-646.Wingender, E. (1990). Transcription regulating proteins and theirrecognition sequences. Critical Reviews in Eukaryotic Gene Expression,1, 11-48.Wysocki, L. J., and Sato, V. L. (1978). Panning for lymphocytes: Amethod for cell selection. Proc. Natl. Acad. Sci. USA, 75, 2844-2848.Xanthopoulos, K. G., Mirkovitch, J., Decker, T., Kuo, C. F., andDarnell, J. E., Jr. (1989). Cell-specific transcriptional control of themouse DNA-binding protein mC/EBP. Proc. Natl. Acad. Sci. USA 86,4117-4121.Yamasaki, K. et al. (1988). Cloning and expression of the humaninterleukin-6 (BSF-2/IFNB2) receptor. Science, 241, 825-828.Young, R. A. and Davis, R. W. (1983). Efficient isolation of genes byusing antibody probes. Proc. Natl. Acad. Sci. USA, 80, 1194-1198.

SUMMARY OF THE INVENTION

The present invention comprises the purification and cloning of HNF-4(hepatocyte nuclear factor 4), a factor originally detected in crudeliver extracts as binding to a DNA element required for thetranscription of the transthyretin (TTR) gene in hepatoma cells (Costaet al., 1989). An amino acid sequence comparison indicates that HNF-4 isa member of the superfamily of steroid/thyroid hormone receptors,ligand-dependent transcription factors which are known to play a role indifferentiation and development (Evans, 1988; Green & Chambon, 1988;Beato, 1989). Whereas all of the other members to date fall into one ofseveral subfamilies based on the nucleotide sequence of theirrecognition sites and the amino acid sequence of the zinc finger region(Umesono & Evans, 1989; Forman & Samuels, 1990), HNF-4 appears torepresent a new subfamily.

More particularly, the present transcription factor is believed to playa regulatory role in the formation of lipid carrying proteins such asApo CIII, as well as possible effects on Apo A1, Apo B, pyruvate kinase,al antitrypsin and glutamine synthetase. The cDNA sequence has beenidentified, and the invention relates to the DNA sequence, recombinantmolecules based thereon, probes, sense and antisense RNA, andappropriately transformed host cells. Diagnostic and therapeuticapplications are likewise contemplated.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1—Purification and Identification of HNF-4

(1A) SDS-PAGE Analysis of HNF-4 Purification from Rat Liver Nuclei.Equivalent fractions of the starting material for each of the five lastchromatographic steps and the peak fraction from the Mono Q column (Fxn38) are shown in a Coomassie blue-stained gel. Oligo #1 and #2 are DNAaffinity columns made from HNF-4P and APF-1 oligonucleotides,respectively. The band in Fxn 38 was estimated to be 54 kD based on therelative mobility of the Markers: 97, 66, 43 and 31 kD, top to bottom.

(1B) Characterization of the Binding Activity of Purified HNF-4 Protein.The protein-DNA complexes from a mobility-shift assay (0.0625 μl Mono QFxn 38, 3 μg BSA, 0.5 μg poly(dl-dc) with seven ³²P-labeledoligonucleotide probes (1 ng) with and without 50-fold excess competitorare shown. APF1, −151=−151 to −130, 4P=HNF4P, 4D=HNF4D as in Table 1.Nonspecific probes are from the mouse TTR promoter: −175=−175 to −151(Costa et al., 1986), HNF3 (−111 to −85, Costa et al., 1989) and c/EBP(−186 kb, site 3, Costa et al., 1988).

(1C) Renaturation of HNF-4 Protein. Fifty nanograms of Mono Q-purifiedHNF-4 was fractionated by SDS-PAGE and the protein eluted from a seriesof gel slices was tested for binding to the APF1 probe (0.5 ng) in amobility-shift assay. Competitor was 50-fold excess unlabeled APF1oligonucleotide. The protein gel lane shown was run in parallel to thedissected lane and is silver stained.

FIG. 2—Characterization of Purified HNF-4

(2A) Footprint: Purified HNF-4 (Fxn 38) was used to footprint bothstrands of the −202 to −70 region of the mouse TTR promoter with copperphenanthroline. “F” and “B” are free and bound probe. “G” designatesprobe cleaved at G residues. The footprinted regions are shown inbrackets; the arrow points to a hypersensitive site.

(2B) Phosphatase and Protease Studies: Purified HNF-4 (Fxn 38) wasincubated at 37□C (−) in the presence of calf intestine alkalinephosphatase (CIP), Protease V8 (V8), or Endoproteinase Lys C (lysC). Thetreated material was divided into four aliquots and tested in themobility-shift assay with the designated probes.

FIG. 3—Nucleotide Sequence of HNF-4 cDNA and Deduced Amino Acid Sequenceof HNF-4 Protein

(3A) Schematic Representation of the Largest HNF-4 Clone, pf7 (SEQID:1). The positions of the peptides obtained from CNBr-cleavage of thepurified protein (pep 1-5, plain lines) and the correspondingoligonucleotide primers which yielded products in PCR (arrows) (notdrawn to scale) are shown. The open reading frame starting from thesecond in-frame methionine (see text) is delineated by the box. Numbersare the nucleotide positions from the beginning of the cDNA. The hatchedarea denotes the region used to probe a rat liver cDNA library for afull length clone. “Zinc finger” refers to the section of similarity tothe steroid hormone receptors.

(3B) Partial Nucleotide Sequence (SEQ ID NO: 1) and Deduced Amino AcidSequence of HNF-4 cDNA. Sequence was obtained from the PCR products, pf7and other cDNA isolates by the dideoxy method (Sanger et al., 1977). Allregions were sequenced from at least two sources and were verified inthe pf7 clone. The underlined amino acid sequences correspond topeptides 1.5. “+1” marks the probable initiator methionine. The bracketmarks the knuckle of the first zinc finger and the (*) denotes the novelasp residue (see text). The sequence has been submitted to GenBank.

FIG. 4—Structural and Sequence Similarity Between HNF-4 Protein andSteroid Hormone Receptors

The primary amino acid sequences of rat HNF-4 was compared to members ofthe receptor superfamily using the FASTA program (Pearson & Lipman,1988). Percentages denote amino acid identity within the zinc finger(Zn++) and ligand binding domains. “Pro” refers to a proline-richdomain. mH2-RIIBP is a mouse major histocompatibility class I regulatoryprotein (Hamada et al., 1989); h c-erbA is the human thyroid hormonereceptor T₃R_(β) (Weinberger et al., 1986); h ER is the human estrogenreceptor (Green et al., 1986); COUP-TT(ear3) is the chicken ovalbuminupstream promoter transcription factor (Wang et al., 1989) and h ear 2is a human v-erbA-related gene (Miyajima et al., 1988).

FIG. 5—In vitro Synthesized HNF-4 Protein Binds to its Recognition Siteas a Dimer

(5A) Schematic Representation of Truncated Forms of HNF-4 ProteinSynthesized in vitro. pf7 DNA (in Bluescript SK(−)) was cut with therestriction enzymes indicated and transcribed in vitro with T3 RNApolymerase. The resulting mRNAs were translated with rabbit reticulatelysate (Promega) in the presence of ³H-leucine. The open box representsthe 3 kb cDNA insert in pf7; the numbers are the nucleotide position ofthe start (ATG) and stop (TAG). The position of the cut site of therestriction enzymes and the length of the polypeptide in amino acids(aa) resulting from translation beginning at nucleotide 59 are given.

(5B) Mobility-Shift Assay of in vitro Synthesized HNF-4 Products.Reactions contained 0.5 ng ³²P-labeled APF1 probe and 2 μg poly(dl-dC)in the presence of 25 ng unlabeled nonspecific (−) (−175 to −151 TTR) orspecific (+) oligonucleotide (APF1) as competitor. Lanes 1-2: purifiedHNF-4 (Fxn 38); lanes 3-12; in vitro translation reactions (2 μl) asdescribed in (A); lanes 13-14: Bovine Mosaid Virus (BMV) RNA added tothe in vitro translation system as a negative control.

(5C) SDS-PAGE of in vitro Synthesized HNF-4 Products. Autoradiogram of a10% gel (treated with Enhance, NEN) containing 1 μl of translationreactions described in (A). The positions of Coomassie-stained markersare shown on the left.

(5D) Mobility-Shift Assay Showing Dimer Formation. pf7 DNA cut with therestriction enzymes indicated was transcribed as in (A). The resultingRNAs were mixed as noted and translated in vitro. The translationreactions were assayed as in (B) in the presence of nonspecificcompetitor. The arrows indicate the complexes formed by heterodimericprotein; the arrow head marks the shift complex normally seen,presumably a homodimer.

FIG. 6—Transcriptional Activation by HNF-4 cDNA

Top: autoradiogram of CAT assay. Bottom: schematic representation ofreporter constructs. Expression vector DNA (0-5.0 μg) containing HNF-4cDNA (the 3 kb insert of pf7) in either the sense or antisense directionwas cotransfected into HeLa cells with a CAT reporter construct (2 μg),either lacking (HIV-CAT) or containing HNF-4 recognition sites(APF1-HIV-CAT). The long terminal repeat (LTR) of the humanimmunodeficiency virus (HIV) served as the basal promoter element.Densitometry of the autoradiogram indicated a 10-15 fold induction byHNF-4 cDNA (lane 2-4 compared to lanes 9-11).

FIG. 7—Limited Tissue Distribution of HNF-4 mRNA

Northern blot analysis of poly(A)+ RNA from different rat and mousetissues using an HNF-4 cDNA fragment as probe (top). A glyceraldehyde3-phosphate dehydrogenase (GAPDH) probe served as a control (bottom).

FIG. 8—HNF-4 Binds to an LF-A1 Site

Mobility-shift assay with either purified (MonoQ, Fxn 38, 0.03 μl ) orin vitro translated HNF-F (Sph 1, FIG. 5, 2 μg poly(dl-dC) and 25 ng ofunlabeled oligonucleotide, either nonspecific (−) (−175 to −151 TTR) orspecific (+) oligonucleotide (APF1, LF-A1 or HNF4P) as competitor.

FIG. 9—HNF-4 does not Significantly Bind ERE, TRE or GRE

Mobility-shift assay using purified HNF-4 (MonoQ, Fxn 38, 0.03 μl) inthe presence of 3 μg BSA, 50 ng poly dl-dC, ³²P-labeled, −151 to −130TTR [probe (0.5 ng) and unlabeled oligonucletides as competitors asindicated: −151−=−151−130 TTR, 4D=NHF4D and ERE, TRE and GRE are theestrogen, thyroid hormone and glucocorticoid response elements (seeTable 1). 0.015 is an unrelated oligonucleotide,5′-GATCCTCGGGAAAGGGAAACCGAAACTGAAGCC-3′ (SEQ ID NO:8. 1, 2 and 3 are50-, 250- and 500-fold molar excess, respectively.

DETAILED DESCRIPTION

In accordance with this detailed description, the following definitionsapply:

Expression control sequence—a DNA sequence that controls and regulatesthe transcription and translation of another DNA sequence.

Operatively linked—a DNA sequence is operatively linked to an expressioncontrol sequence when the expression control sequence controls andregulates the transcription and translation of that DNA sequence. Theterm “operatively linked” includes having an appropriate start signal(e.g., ATG) in front of the DNA sequence to be expressed and maintainingthe correct reading frame to permit expression of the DNA sequence underthe control of the expression control sequence and production of thedesired product encoded by the DNA sequence. If a gene that one desiresto insert into a recombinant DNA molecule does not contain anappropriate start signal, such a start signal can be inserted in frontof the gene.

Antibody—an immunoglobulin molecule or functional fragment thereof, suchas Fab, F(ab′)₂ or dAB. An antibody preparation is reactive for aparticular antigen when at least a portion of the individualimmunoglobulin molecules in the preparation recognize (i.e., bind to)the antigen. An antibody preparation is nonreactive for an antigen whenbinding of the individual immunoglobulin molecules in the preparation tothe antigen is not detectable by commonly used methods.

Standard hybridization conditions—salt and temperature conditionssubstantially equivalent to 5×SSC and 65° C. for both hybridization andwash.

-   -   DNA sequences—The DNA sequences of this invention refer to DNA        sequences prepared or isolated using recombinant DNA techniques.        These include cDNA sequences, DNA sequences isolated from their        native genome, and synthetic DNA sequences. The term as used in        the claims is not intended to include naturally occurring DNA        sequences as they exist in nature.

HNF-4 (hepatocyte nuclear factor 4) is a protein enriched in liverextracts that binds to sites required for the transcription of thetransthyretin (TTR) and apolipoprotein CIII (apoCIII) genes (Costa etal., 1989; Costa et al., 1990; Leff et al., 1989). HNF-4 protein (54 kD)has been purified and a cDNA clone isolated encoding the protein. HNF-4is a member of the steroid hormone receptor superfamily with an unusualamino acid in the conserved “knuckle” of the first zinc finger (DGCKG).This and the fact that HNF-4 does not bind significantly to estrogen,thyroid hormone or glucocorticoid response elements indicate that HNF-4may represent a new subfamily. HNF-4 binds to its recognition site as adimer and activates transcription in a sequence-specific fashion innonhepatic (HeLa) cells. HNF-4 mRNA is present in kidney and intestineas well as liver but is absent in other tissues. DNA binding datasuggest that HNF-4 could be identical to liver factor A1 (LF-A1), afactor previously shown to regulate the transcription of the α-1antitrypsin, apolipoprotein A1 and pyruvate kinase genes.

As used herein, the word “ligand” means a substance which binds to areceptor, such as a hormone or growth substance. Inside a cell theligand binds to a receptor protein, thereby creating a ligand/receptorcomplex, which in turn can bind to an appropriate hormone responseelement. Single ligands may have multiple receptors. For example, boththe T₃R_(α) and the T₃R_(β) bind thyroid hormone such as T₃. The ligandcan be an agonist or an antagonist.

As used herein, the word “operative”, in the phrase “operative hormoneresponse element functionally linked to a ligand-responsive promoter andan operative reporter gene”, means that the respective DNA sequences(represented by the terms “hormone response element”, “ligand-responsivepromoter” and “reporter gene”) are operational, i.e., the hormoneresponse element can bind with the DNA-binding domain of receptorprotein (either wild-type or chimeric), the ligand-responsive promotercan control transcription of the reporter gene (upon approrpiateactivation by a HRE/-receptor protein/ligand complex) and the reportergene is capable of being expressed in the host cell. The phrase“functionally linked” means that when the DNA segments are joined, uponappropriate activation, the reporter gene (.e.g., CAT or luciferase)will be expressed. This expression occurs as the result of the fact thatthe “ligand responsive promoter” (which is downstream from the hormoneresponse element, and “activated” when the HRE binds to an appropriateligand-/receptor protein complex, and which, in turn then “controls”transcription of the reporter gene) was “turned on” or otherwiseactivated as a result of the binding of a ligand-/receptor proteincomplex to the hormone response element.

As used herein, the phrase “DNA-binding domain” of receptors refers tothose portions of the receptor proteins (such as glucocorticoidreceptor, thyroid receptor, mineralocorticoid receptor, estrogen-relatedreceptor and retinoic acid receptor) that bind to HRE sites on thechromatin DNA. The boundaries for these DNA-binding domains have beenidentified and characterized for the steroid hormone superfamily. SeeFIG. 8; also see Giguere et al. (1986); Hollenberg et al. (1987); Greenand Chambon (1987); and Miesfield et al. (1987), Evans (1988).

The present transcription factor is believed to play a regulatory rolein the formation of lipid carrying proteins such as Apo CIII, as well aspossible effects on Apo A1, Apo B, pyruvate kinase, al antitrypsin andglutamine synthetase. The cDNA sequence has been identified, and theinvention relates to the DNA sequence, recombinant molecules basedthereon, probes, sense and antisense RNA, and appropriately transformedhost cells. Diagnostic and therapeutic applications are likewisecontemplated.

Of particular interest herein is the APF1 receptor and its gene, sincethese structures are useful for assessing the activity of drugs.

Numerous epidemiological studies have shown that altered plasmalipoprotein levels are associated with coronary heart disease risk.Elevated low-density lipoprotein (LDL) levels and decreased high-densitylipoprotein (HDL) levels are associated with increased coronary heartdisease. Studies conducted in many laboratories over the last 30 yearshave defined a rather complex set of events that determine plasmalipoprotein levels.

Apolipoprotein CIII is a constituent of VLDL and HDL and comprises −50%of VLDL protein and 2% of HDL protein. Human plasma apoCIIIconcentrations are in the range of 0.12-0.14 mg/ml. ApoCIII is aglycoprotein containing 1 mol each of galactose, galactosamine, andeither 0, 1, or 2 mol of sialic acid. The three resultant isoproteinsrecognizable by isoelectric focusing are designated CIII-0, CIII-1, andCIII-2 and comprise 14, 59, and 27% of plasma apo CIII, respectively. Invitro apoCIII has been shown to inhibit the activities of bothlipoprotein lipase and hepatic lipase. ApoCIII has also been shown todecrease the uptake of lymph chylomicrons by the perfused rat liver.These in vitro studies suggest that apo CIII might delay catabolism oftriglyceride-rich lipoproteins. Recently, hypertriglyceridemic subjectswere shown to have circulation lipoprotein and nonlipoprotein inhibitorsof lipoprotein lipase. The lipoprotein-associated inhibition correlatedbest with apo CIII concentration. In the same study, apoCIII was shownto be a noncompetitive inhibitor of the activity of partially purifiedlipoprotein lipase. In addition, patients with combined apo A-I andapoCIII deficiency were shown to have low plasma triglyceride levels,and in vivo studies showed that they rapidly convert VLDL to LDL. Invitro lipolysis of their VLDL was inhibited by added apoCIII. Thus, itappears that primary abnormalities in the quantity or quality of apoCIIImay affect plasma triglyceride levels, and the physiological role ofapoCIII may be in the regulation of the catabolism of triglyceride-richlipoproteins. Functional domains of apoCIII have been demonstrated. TheCOOH-terminal 39 amino acids bind phospholipid, whereas the NH₂-terminal40 amino acids do not. Synthesis of apoCIII is mainly in liver and to alesser degree in intestine.

It is apparent that there is a wide variety of medical uses for agonistsand antagonists of HNF-4 and apoCIII. For example, diseases involvingthe cardiovascular system, such as atherosclerotic heart disease,hyperlipidemia and arteriosclerosis can be treated by interfering withthe deposition of VLDL and cholesterol in the vessels.

Similarly, liver disease involving the presence of excessive lipidlevels can be treated.

Other disease conditions in which the ligands to HNF-4 andagonists/antagonists to apoCIII will be apparent to those skilled in themedical arts, using such compounds in art-recognized doses.

Likewise, conditions such as obesity may be treated in this manner.

Ligands to HNF-4 may be evaluated which have pharmaceutical properties.One assay format which can be used which employs two genetic constructs.One is typically a plasmid that continuously expresses the receptor ofinterest when transfected into an appropriate cell line. CV-1 cells aremost often used. The second is a plasmid which expresses a reporter,e.g., luciferase under control of a receptor/ligand complex. Forexample, if a compound which acts as a ligand for HNF-4 is to beevaluated, one of the plasmids would be a construct that results inexpression of the HNF-4 receptor in an appropriate cell line, e.g., theCV-1 cells. The second would possess a promoter linked to the luciferasegene in which an HNF-4 response element is inserted. If the compound tobe tested is an agonist for the HNF-4 receptor, the ligand will complexwith the receptor and the resulting complex binds the response elementand initiates transcription of the luciferase gene. In time the cellsare lysed and a substrate for luciferase added. The resultingchemiluminescence is measured photometrically. Dose response curves areobtained and can be compared to the activity of known ligands.

Other reporters than luciferase can be used including CAT and otherenzymes.

Viral constructs can be used to introduce the gene for the receptor andthe reporter. The usual viral vector is an adenovirus. For furtherdetails concerning this preferred assay, see U.S. Pat. No. 4,981,784issued Jan. 1, 1991 hereby incorporated by reference, and Evans et al.,WO88/03168 published on May 5, 1988, also incorporated by reference.

HNF-4 antagonists can be identified using this same basic “agonist”assay. A fixed amount of an antagonist is added to the cells withvarying amounts of test compound to generate a dose response curve. Ifthe compound is an antagonist, expression of luciferase is suppressed.

The APF1 gene can also be incorporated into the assay described above.Agonist ligands can be screened by the continuous expression ofreceptors, and by evaluating ligand binding to the receptors, andthereafter quantitating the production of the reporter.

Genes for chimeric receptors can be used in the assay system. Thesechimeric receptors have hybrid functional characteristics based on the“origin” of the “parental” DNA-binding and ligand-binding domainsincorporated within the chimeras. For example, if the DNA-binding domainin the chimeric receptor is a retinoic acid receptor DNA-binding domain(i.e., is obtained from wild-type retinoic acid receptor or is a mutantthat contains the functional elements of retinoic acid DNA-bindingdomain), then the chimera will have DNA-binding propertiescharacteristic of a retinoic acid receptor. The same is true of theligand-binding domain. If the ligand-binding domain in the chimericreceptor binds to thyroid hormone, then the chimera will haveligand-binding properties characteristic of a thyroid hormone receptor.Most often this is done for a so-called orphan receptor, i.e., one wherethe natural ligand is unknown. The chimerics usually constructed areones in which the ligand binding domain of a gene for a known receptor,for example, a glucocorticoid receptor, is replaced by the ligandbinding domain of the orphan. The resulting construct generates areceptor with the ligand binding domain of the orphan and the DNAbinding domain of the glucocorticoid receptor. Thus, the receptor can beused to control a glucocorticoid controlled gene. Ligands to the orphanare thereby screened in an otherwise well developed system. The HNF-4gene can be used in this manner.

Genes for the receptors in expression systems can also be employed whichare capable of producing large amounts of a receptor which can bepurified and used in binding assays. These assays are done in acompetitive format in which the suspect ligand competes for receptorwith a quantity of a known, labeled ligand. These assays can be used toconfirm that the ligand does bind the receptor, and as furtherconfirmation that the results of the cis/trans assay are not artifacts.The systems used to express large amounts of receptors include virallyinfected cells in which the gene for the receptor is introduced by aviral construct by infection rather than by plasmid transfection.Adenoviruses are preferred. Also, a yeast based system can be used wherethe receptor gene is inserted into a plasmid suitable for yeastexpression.

The gene for HNF-4 receptors may be inserted, for example, into a viralconstruct, and the viral vector with HNF-4 receptor genes can be used tooverexpress receptors for HNF-4 as well as in the convection form of theassay noted above.

Expression of recombinant DNA molecules according to this invention mayinvolve post-translational modification of a resultant polypeptide bythe host cell. For example, in mammalian cells expression might include,among other things, glycosylation, lipidation or phosphorylation of apolypeptide, or cleavage of a signal sequence to produce a matureprotein. Accordingly, as used herein, the term HNF-4 encompassesfull-length polypeptides and modifications or derivatives thereof, suchas glycosylated versions of such polypeptides, mature proteins,polypeptides retaining a signal peptide, truncated polypeptides havingcomparable biological activity, and the like.

mRNA can be isolated from cells expressing HNF-4, and used to create aCDNA library. Many methods are known for isolating mRNA and forproducing cDNA from it. (See, e.g., Gubler and Hoffman, 1983 andManiatis et al., 1982.)

The CDNA is then inserted into an appropriate vector. The vector pcDM8,described by Brian Seed (Seed, 1987) is representative. This plasmid hasseveral advantages including a high copy number in E.coli, a eukaryoticpromoter, and high level of expression in transient expression systemssuch as COS 7 cells. However, several other vector systems areavailable. (See, e.g., Cate et al., 1986.)

After constructing a cDNA library, the next step is to isolate from itclones containing HNF-4 cDNA sequences. There are currently many ways toisolate cDNA for a differentially expressed mRNA. These include, forexample, (1) plus/minus screening with labeled cDNA; (2) production ofsubtracted cDNA libraries; and (3) screening with subtractive cDNAprobes. (Davis, 1986; Sargent, 1987; Davis et al., 1985, Hedrick et al.,1984; and Duguid et al., 1988.)

Different techniques can be used to identify clones that contained cDNAfor HNF-4 sequences. In a first method, clones can be tested forexpression of HNF-4 activity in an appropriate eukaryotic expressionsystem. One can use a variety of direct expression techniques, includingantibody screening of fusion proteins encoded by cDNA cloned in λGTll(Young and Davis, 1983; Young and Davis, 1984); or activity assay ofoocyte-conditioned media after injection of mRNA from cloned cDNA, orfrom plasmid or phage DNA carrying SP6/T7 promoters. Alternatively, onecan make libraries in plasmid, phage, and cosmid vectors containing avariety of promoter, selection and replication elements. Animal cellsmay be transfected with the library for transient or stable expression.Transfection can be accomplished by a variety of methods. For transientexpression, investigators have used spheroplast fusion, DEAE dextran,and electroporation. For stable expression they have used calciumphosphate, spheroplast fusion, and electroporation.

Until recently, identification of cloned molecules by direct expressionhas required sensitive assays and has been restricted to lymphokines.However, cDNA cloning of single-chain cell-surface molecules inefficient transient expression vectors (see, e.g., Seed and Aruffo, 1987and Seed, 1987), either by antibody “panning” technology (Wysocki andSato, 1978) or by identification of functional molecules by FACS(Yamasaki et al., 1988), has expanded the range of cloned molecules thatone can identify by direct expression.

Genomic DNA sequences, including transcriptional promoters, for HNF-4can be isolated by screening genes. A human genomic library with³²P-labeled probes derived from the coding regions of the HNF-4 DNAsequences. This may yield clones that contain portions of theuntranscribed and untranslated regions of the HNF-4 gene.

Transcriptional promoters have a number of uses. First, they are usefulto construct vectors which can be used to induce expression of HNF-4.Such vectors may be useful, for example, in gene transfer assays,wherein the inducible promoter is positioned so that it drivestranscription of a reporter gene such as chloramphenicolacetyltransferase (CAT), beta-galactosidase, luciferase, etc. Thisconstruct can then be introduced transiently or in stable form into anappropriate mammalian cell line. Potential inhibitors or stimulators ofinduction can then be assayed by measuring their effect on induction byany or all of the inducers listed above.

Hybridomas producing monoclonal antibodies which recognize HNF-4 canalso be produced.

Investigators are also exploring radioimmunotherapy and immunotoxintherapy. Radioimmunotherapy involves the use of radioimmunoconjugates inwhich nuclides such as ¹²⁵I, ⁹⁰Y, ¹⁸⁶Re and the like are bound toantibodies recognizing a particular surface antigen. Immunotoxins areantibodies conjugated with cell toxins, such as Pseudomonas exotoxin andthe like. Upon injection, these conjugated antibodies target the toxicagents to cells expressing the antigen. In accordance with thisinvention, radioactive markers, nuclides and cellular toxins may beconjugated with HNF-4, or antibodies recognizing HNF-4, target cellsexpressing HNF-4 or ligands thereto.

An alternative method for isolating HNF-4 would employfluorescent-antibody labeling. In this method, HNF-4 expressing cellsare incubated with Moabs (monoclonal antibodies) and then the Moabs arelabeled with, e.g., fluorescently tagged anti-mouse antibody. Cellsbinding the fluorescent antibodies may then be sorted with afluorescence activated cell sorter (FACS). The DNA from the sorted cellsmay be used to transform a bacterial host such as E. coli. DNA from theresulting colonies may then be used to transform a bacterial host suchas E. coli. DNA from the resulting colonies may then be used totransfect an appropriate cell line, and this procedure may be repeateduntil a single expressing clone is identified.

An expression library may also be created in E. coli. For example, a λZAP® (Stratagene)/HL-60 library may be constructed and used to expressthe inserted DNA in E. coli. After plating, the plaques can be directlyscreened with, e.g., radioactively labeled monoclonals (Young and Davis,1983 and Young and Davis, 1984). The plaques to which the monoclonalsbind can be picked and the DNA insert isolated from them.

Another method to identify HNF-4 ligands, not based on antibodyrecognition, is to transfect COS 7 cells with an approrpiate library,that may be subtracted, and then pan them directly into HNF-4 expressingcells. Once again, multiple rounds of panning may be required to enrichthe library sufficiently to isolate the pertinent clones.

Another technique for isolating the DNA sequences involves screening acDNA library with oligonucleotide probes. If sufficient HNF-4 protein ispurified, for example by affinity chromatography using immobilizedantibody, one may determine a partial amino acid sequence and synthesizeoligonucleotide probes that correspond to at least a portion of thegene. These probes may then be used to screen the cDNA library.Alternatively, the oligonucleotides may be used as primers to generatelong probes to be used in screening the library for genes.

Several uses for HNF-4 DNA sequences and molecules are contemplated asbeing part of the present invention. First, one may use HNF-4 to producemonoclonal antibody preparations that are reactive for these molecules.The Moabs may be used diagnostically or in turn as therapeutic agents toinhibit HNF-4 binding.

Second, one may use a soluble form of HNF-4 or fragments thereof as abinding inhibitor. The HNF-4 peptides would bind to the HNF-4 ligandsand the HNF-4 ligand would bind to HNF-4 receptors. Both methods wouldthereby inhibit HNF-4 binding.

To produce recombinant soluble HNF-4 ligand, one could, for example,alter a DNA encoding those molecules to eliminate the transmembraneregion. Thus, DNAs for soluble molecules would include all or part ofthe extracellular domain, perhaps attached to the cytoplasmic domain.This approach has already been validated using soluble CD4, the surfaceprotein on T-cells that binds to the AIDS virus (Fisher et al., 1988).This approach also avoids the problems of antibody therapy, since thepolypeptides used would be less likely to induce an immune response.

One problem investigators have encountered with soluble recombinantmolecules is a short in vivo plasma half-lie (Capon et al., 1989).Because such molecules are quickly cleared from the system, large dosesor frequent injections are necessary to have a therapeutic effect.Therefore, investigators have sought methods to increase the half-lifeof soluble molecules. A potential solution is to link the solublemolecule to another molecule known to have a longer half-life in theblood stream. Due to their long half life, immunoglobulin molecules arepromising candidates. Capon et al. (1989) have described the linking ofsoluble CD4 to an immunoglobulin molecule using recombinant DNAtechniques. In this approach, one replaces the variable region of animmunoglobulin molecule with the soluble protein, forming aprotein/immunoglobulin fusion protein.

It is expected that the recombinant soluble immunoglobulin fusionproteins will have greater plasma half-life than the soluble proteinalone. Such fusion proteins are preferably produced with recombinantconstructs, fusing a DNA sequence encoding the soluble molecule to a DNAsequence encoding the constant domain of an immunoglobulin molecule. Therecombinant DNA may then be expressed in an approrpiate host cell,preferably an animal cell, to produce the fusion protein.

Immunoglobulin fusion proteins have another advantage. Becauseimmunoglobulin molecules are normally bivalent (i.e., they have twobinding sites), an immunoglobulin fusion protein would have two HNF-4sand so, two ligand binding sites. Therefore; one would expect them tohave greater affinity or avidity for cells displaying HNF-4 ligands.

Third, one may use molecules binding to HNF-4 receptors (such asanti-HNF-4 antibodies, or markers such as the ligand or fragments of it)to detect the presence of disease. This involves, for example, making amolecule detectable by fluorescence or radioactivity, administering itto a patient and determining where in the body it accumulates. In thisway one could also identify the type of disease.

Fourth, if HNF-4 binds to its ligand through a carbohydrate moiety orsome other post-translational modification, one could use HNF-4 toidentify the carbohydrate on the HNF-4 ligand to which it is bound.

Fifth, one could use HNF-4 as part of a system to screen small moleculesfor inhibitors. For example, one could create an assay system in whichsmall molecules are tested for the ability to inhibit the interactionbetween HNF-4 and ligands thereto. Small molecule inhibitors identifiedin this way would provide drug candidates.

Sixth, one could use these molecules to identify endogenous proteinsthat inhibit HNF-4.

Seventh, one can generate fusion proteins. It is known that proteins arecomposed of several structural domains (Simmons et al., 1988). DNAsequences encoding various domains of each protein are fused using, forexample, the genetic fusion techniques described for makingimmunoglobulin fusion proteins. The domains chosen are those having theability to bind to ligands and HNF-4. Domains binding to known ligandswould be preferable. The polypeptides produced on expression of theseDNA sequences are useful because they would block adhesion of any cellhaving a ligand to either the HNF-4 receptor, the ligand or both.

Finally, one could use HNF-4 and HNF-4 ligand DNA sequences to producenucleic acid molecules that intervene in HNF-4 or HNF-4 ligandexpression at the translational level. This approach utilizes antisensenucleic acid and ribozymes to block translation of a specific mRNA,either by masking that mRNA with an antisense nucleic acid or cleavingit with a ribozyme. These methods will also be useful in treatingdisease conditions.

Antisense nucleic acids are DNA or RNA molecules that are complementaryto at least a portion of a specific mRNA molecule. (See Weintraub, 1990;Marcus-Sekura, 1988.) In the cell, they hybridize to that mRNA, forminga double stranded molecule. The cell does not translate an mRNA in thisdouble-stranded form. Therefore, antisense nucleic acids interfere withthe expression of mRNA into protein. Oligomers of about fifteennucleotides and molecules that hybridize to the AUG initiation codonwill be particularly efficient, since they are easy to synthesize andare likely to pose fewer problems than larger molecules when introducingthem into HNF-4-producing cells. Antisense methods have been used toinhibit the expression of many genes in vitro (Marcus-Sekura, 1988;Hambor et al., 1988).

Ribozymes are RNA molecules possessing the ability to specificallycleave other single stranded RNA molecules in a manner somewhatanalogous to DNA restriction endonucleases. Ribozymes were discoveredfrom the observation that certain mRNAs have the ability to excise theirown introns. By modifying the nucleotide sequence of these RNAS,researchers have been able to engineer molecules that recognize specificnucleotide sequences in an RNA molecule and cleave it (Cech, 1988.).Because they are sequence-specific, only mRNAs with particular sequencesare inactivated.

Investigators have identified two types of ribozymes, Tetrahymena-typeand “hammerhead”-type. (Hasselhoff and Gerlach, 1988) Tetrahymena-typeribozymes recognize four-base sequences, while “hammerhead”-typerecognize eleven- to eighteen-base sequences. The longer the recognitionsequence, the more likely it is to occur exclusively in the target mRNAspecies. Therefore, hammerhead-type ribozymes are preferable toTetrahymena-type ribozymes for inactivating a specific mMA species, andeighteen base recognition sequences are preferable to shorterrecognition sequences.

The DNA sequences described herein may thus be used to prepare antisensemolecules against, and ribozymes that cleave, mRNAs for HNF-4 and HNF-4ligands.

Antisense molecules and ribozymes may be used in methods to treatdisease by introducing into cells molecules that interfere with theexpression of HNF-4. Since therapeutic agents can be delivered easily byintravenous injection, hepatocytes are attractive targets for suchtherapies, provided the antisense molecules or ribozymes can bedelivered effectively.

Investigators have suggested two approaches which could be used todeliver these molecules to target cells. The first involves transfectingthe target cell with a vector that expresses the anti-HNF-4 antisensenucleic acid or the HNF-4-specific ribozymes as an mRNA molecule (Hamboret al., supra). While this approach is very useful when dealing withcell lines in vitro, it may not be as effective in vivo. A secondapproach that is more promising for in vivo delivery involves loadingliposomes with anti-HNF-4 antisense molecules, HNF-4-specific ribozymesor vectors which express them. These liposomes could also containmonoclonal antibodies to direct the liposome to the site of disease.

Another feature of this invention is the expression of the DNA sequencesdisclosed herein. As is well known in the art, DNA sequences may beexpressed by operatively linking them to an expression control sequencein an appropriate expression vector and employing that expression vectorto transform an appropriate unicellular host.

Such operative linking of a DNA sequence of this invention to anexpression control sequence, of course, includes, if not already part ofthe DNA sequence, the provision of an initiation codon, ATG, in thecorrect reading frame upstream of the DNA sequence.

A wide variety of host/expression vector combinations may be employed inexpressing the DNA sequences of this invention. Useful expressionvectors, for example, may consist of segments of chromosomal,non-chromosomal and Synthetic DNA sequences. Suitable vectors includederivatives of SV40 and known bacterial plasmids, e.g., E.coli plasmidscol El, pCR1, pBR322, pMB9 and their derivatives, plasmids such as RP4;phage DNAS, e.g., the numerous derivatives of phage λ, e.g., NM989, andother-phage DNA, e.g., M13 and Filamentous single stranded phage DNA;yeast plasmids such as the 2μ plasmid or derivatives thereof; vectorsuseful in eukaryotic cells, such as vectors useful in insect ormammalian cells; vectors derived from combinations of plasmids and phageDNAS, such as plasmids that have been modified to employ phage DNA orother expression control sequences; and the like.

Any of a wide variety of expression control sequences—sequences thatcontrol the expression of a DNA sequence operatively linked to it—may beused in these vectors to express the DNA sequences of this invention.Such useful expression control sequences include, for example, the earlyand late promoters of SV40 or adenovirus, the lac system, the trpsystem, the TAC or TRC system, the major operator and promoter regionsof phage λ, the control regions of fd coat protein, the promoter for3-phosphoglycerate kinase or other glycolytic enzymes, the promoters ofacid phosphatase (e.g., Pho5), the promoters of the yeast α-matingfactors, and other sequences known to control the expression of genes ofprokaryotic or eukaryotic cells or their viruses, and variouscombinations thereof.

A wide variety of unicellular host cells are also useful in expressingthe DNA sequences of this invention. These hosts may include well knowneukaryotic and prokaryotic hosts, such as strains of E. coli,Pseudomonas, Bacillus, Streptomyces, fungi such as yeasts, and animalcells, such as CHO, R1.1, B-W and L-M cells, African Green Monkey kidneycells (e.g., COS 1, COS 7, BSC1, BSC40, and BMT10), insect cells (e.g.,Sf9), and human cells and plant cells in tissue culture.

It will be understood that not all vectors, expression control sequencesand hosts will function equally well to express the DNA sequences ofthis invention. Neither will all hosts function equally well with thesame expression system. However, one skilled in the art will be able toselect the proper vectors, expression control sequences, and hostswithout undue experimentation to accomplish the desired expressionwithout departing from the scope of this invention. For example, inselecting a vector, the host must be considered because the vector mustfunction in it. The vector's copy number, the ability to control thatcopy number, and the expression of any other proteins encoded by thevector, such as antibiotic markers, will also be considered.

In selecting an expression control sequence, a variety of factors willnormally be considered. These include, for example, the relativestrength of the system, its controllability, and its compatibility withthe particular DNA sequence or gene to be expressed, particularly asregards potential secondary structures. Suitable unicellular hosts willbe selected by consideration of, e.g., their compatibility with thechosen vector, their secretion characteristics, their ability to foldproteins correctly, and their fermentation requirements, as well as thetoxicity to the host of the product encoded by the DNA sequences to beexpressed, and the ease of purification of the expression products.

Considering these and other factors a person skilled in the art will beable to construct a variety of vector/expression control sequence/hostcombinations that will express the DNA sequences of this invention onfermentation or in large scale animal culture.

Antibodies against HNF-4 and ligands thereto will make possible anothermethod for isolating other ligands. The method takes advantage of anantibody characteristic known as idiotypy. Each antibody contains aunique region that is specific for an antigen. This region is called theidiotype. Antibodies, themselves, contain antigenic determinants; theidiotype of an antibody is an antigenic determinant unique to thatmolecule. By immunizing an organism with antibodies, one can raise“anti-antibodies” that recognize them, including antibodies thatrecognize the idiotype. Antibodies that recognize the idiotype ofanother antibody are called anti-idiotypic antibodies. Someanti-idiotypic antibodies mimic the shape of the original antigen thatthe antibody recognizes and are said to bear the “internal image” of theantigen (Kennedy, 1986). When the antigen is a ligand, certainanti-idiotype antibodies that bind to receptors including the receptorfor insulin, angiotensin II, adenosine I, β-adrenalin, and rat brainnicotine and opiate receptors (Carisson and Glad, 1989).

Taking advantage of this phenomenon, other HNF-4 ligands may be isolatedusing anti-idiotypic antibodies. Anti-idiotypes may be used to screenfor molecules binding to the original antigen.

EXPERIMENTAL PROCEDURES

Extract preparation and chromatography were carried out at 4° C.

Preparation of Rat Liver Nuclear Extract

Crude rat liver nuclei extracts were prepared using the method of Gorskiet al. (1986) modified as follows: approximately 50 gm of tissue wereremoved from 3 to 4 freshly sacrificed male rat (Sprague-Dawley, about20 weeks old), homogenized in 30 ml of buffet A (10 mM HEPES pH 7.9, 25mM KCl, 0.15 mM spermine, 0.5 mM spermidine, 1.0 mM EGTA, 1.0 mM EDTA, 1mM dithiothreitol (DTT), 0.32 M sucrose), dounced 5 to 7 times (pestleA) and diluted with two volumes of Buffer b (as A except 2 M sucrose).27 ml of the homogenate were layered on a 10-ml cushion of Buffer B andcentrifuged in a Beckman SW27 rotor at 15 krpm for 45 min. The pelletednuclei were rinsed once in buffer C (as buffer A except 20% glycerol inplace of sucrose), dounced 5 times (pestle B) and brought to 0.41M KClwith buffer D (as C except 1M KCl ). The protein was extracted by gentlerocking at 4° C. for 45 minutes. The chromatin was pelleted bycentrifugation at 180,000×g for 45 min. and the supernatant (crudenuclear extract, 3.5-5.0 mg/ml protein) was frozen immediately in liquidN₂ and stored at −80° C. DTT and protease inhibitors(phenylmethyl-sulfanyl fluoride, 0.5 mM; benzamidine HCl, 1 mM;leupeptin, 0.5 μg/ml; pepstatin, 1 μg 7 ml) were added to all buffersjust prior to use.

Mobility-shift Assay and Purification of HNF-4

Gel mobility-shift (DNA binding) assays (Fried & Crothers, 1981) werecarried out in 15 μl reactions in shift buffer (20 mM HEPES (pH 7.9), 40mM KCl, 2 mM MgCl₂, 1 mM DTT, 0.5 mM EGTA, 4% Ficoll) and contained 1-2μl protein extract and 0.5 ng double-stranded oligonucleotide probelabeled with ³²P by Kenow. Reactions were incubated at room temperaturefor 20 minutes. Poly (dl-dC), oligonucleotide competitor and bovineserum albumin (BSA) were added as indicated. Protein-bound DNA complexes(5 μl of shift reaction) were separated from free probe byelectrophoresis on an 8% polyacrylamide gel in 25 mM Tris-borate, 0.25mM EDTA at 4° C.

Chromatography fractions were assayed by the mobility-shift assay usingeither the APF-1 or HNF4P oligonucleotide as probe. Crude nuclearextract (up to 300 mg) was applied to a 60 ml heparin agarose (Sigma,Type 1) column equilibrated in buffer E (20 mM HEPES pH 7.9, 10%glycerol, 1 mM DTT, 0.1 mM EDTA, 0.1 Mm EGTA) containing 150 mM KCl. Thecolumn was developed with a 400 ml linear gradient from 0.2 to 0.8 MKCl. Fractions with HNF-4 activity (0.50-0.55 M KCl ) were pooled,precipitated with ammonium sulfate (300 mg/ml final), dissolved inbuffer F (as buffer E but with 0.05% Nonidet P-40 (NP-40)) containing100 mM NaCl, dialyzed and loaded onto a 240 ml Sephacryl S300(Pharmacia) column. Active fractions, eluting just after the voidvolume, were loaded onto a 5 ml double-stranded DNA cellulose (Sigma)column equilibrated in buffer F/100 mM NaCl. The column was developedwith a three-step gradient: 150 mM, 300 mM and 1 M NaCl. Activefractions (eluting at 300 mM NaCl) were diluted to 100 mM NaCl and poly(dl-dC) and sonicated, denatured salmon sperm DNA were added to 10 μg/mleach. After 10 minutes on ice, the sample was loaded onto a 2 ml HNF4Poligonucleotide affinity column prepared as in Kadonaga and Tjian (1986)and equilibrated in buffer F/100 mM NaCl. The column was developed witha 20 ml linear gradient from 0.1 to 1.0 M NaCl. Active fractions,eluting at 0.18-0.3 M NaCl, were diluted to 0.1 M NaCl, supplementedwith poly (dl-dC) and salmon sperm DNA to 3 μg/ml each and passed over a2 ml APF1 oligonucleotide affinity column as described above. The HNF-4binding activity, eluting at 0.25 to 0.5 M NaCL, was dialyzed againstbuffer T (as buffer F but with 20 mM Tris HCl pH 8.0 and 20% glycerol)containing 100 mM NaCl and loaded onto a FPLC Mono Q HR 5/5 (Pharmacia)column. The column was developed with a linear gradient from 0.1 to 1.0M NaCl. The peak fraction in one preparation (fraction 38) eluted atabout 0.42 M NaCl. Purified HNF-4 refers to material passed over allfive columns.

Renaturation of HNF-4

Approximately 50 ng of purified HNF-4 (based on binding activity to APF1oligonucleotide) were mixed with SDS sample buffer, heated for 15 min.at 72° C. and fractionated on a 12.5 cm 10% SDS-polyacrylamide gel(Laemmli, 1970) pre-run with 0.1 mM sodium thioglycolate. Gel sliceswere cut out and the protein was eluted and renatured essentially asdescribed by Briggs et al. (1986) except that 0.1 mg/ml BSA was added tothe elution buffer and buffer G (as buffer E but with 0.1% NP-40)containing 100 mM NaCl and 3.5 mM MgCl₂ and 6M guanidine-HCl were usedfor renaturation. 5 μl of 35 μl recovered material was used in themobility shift assay (0.05 μg poly (dl-dC).

DNA Footprinting, Phosphatase and Protease Studies

A 137-bp DNA fragment containing −202 to −70 of the mouse TTR promoter(see Costa et al., 1986) was labeled with ³²P by filling in with Klenoweither at a BamH 1 site (7 bp from −202) or at an Xba 1 site (−70).Purified HNF-4 (enough to shift 2 ng of APF1 oligonucleotide) wasincubated in a 30 μl shift reaction with 10 ng of the −202/−70 TTR probein the absence of poly dl-dC and electrophoresed on a 5% polyacrylamidegel. After treating the gel with 1,10-phenanthroline copper ion asdescribed in Kuwabara and Sigman (1987), the bound and free probes(identified by autoradiography of the wet gel) were cut out, embedded inagarose and the DNA was recovered by electroelution onto DEAE membrane(NA-45 (Schleicher & Schuell)). The cleaved probes were analyzed on an 8M urea/10% polyacrylamide gel.

For the phosphatase reaction, purified HNF-4 (MonoQ fxn 38, 4 ng) wasincubated for 20 min. at 37° C. in a 20 μl, reaction either with orwithout calf intestine alkaline phosphatase (CIP, 2.5 μl at 1 U/μlBoehringer Mannheim) in 0.25×shift buffer lacking KCl and EGTA butcontaining 0.005% NP-40 and 0.25 μg/ul BSA. The reaction without enzymecontained 2.5 μl of the CIP storage buffer (30 mM triethanolamine pH7.6, 3 M NaCl, 1 mM MgCl₂, 0.1 mM ZnCl₂). For the protease reactions,purified HNF-4 (fxn 38, 62.5 ng) was incubated for 1.5 hours at 37° C.in a 10 μl reaction with Protase V8 (5 ng) or Endoproteinase LysC (5 ng)(both from Boehringer Mannheim) in 0.5×butter T containing 100 mM NaCl.One-fifth of each reaction was tested in the mobility-shift assay (BSAat 3 μg/15 μl reaction, no poly (dl-dC) with each of four ³²P-labeledoligonucleotide probes (APF1, −151 to −130, HNF4P, HNF4D).

Cyanogen Bromide Cleavage and Protein Sequencing

Approximately 10 μg (200 pmoles) of purified HNF-4 (fxn 38) was broughtto 1.3 M guanidine HCL (ultra pure, ICN) and 0.03% β-mercaptoethanol(Sigma) and loaded onto a reverse-phase HPLC column (Aquaporebutyl30×2.1 mm, 7 μm, Brownlee labs) equilibrated in buffer H (5% 1-propanolin 10 mM trifluoroacetic acid, TFA). The column was developed with a 9ml-gradient from 5% to 59% 1-propanol in 10 mM TFA at a flow rate of0.15 ml/min. Fractions containing HNF-4 (47% to 50% propanol) werepooled, dried, and treated with 5 μg/ml CNBr in 50% formic acid for 24hours. The CNBr-generated peptides were separated by HPLC using theconditions given above. Fractions containing peptides were eithersequenced directly on an Applied Biosystems gas phase (Model 470)sequenator (pep 1, pep 2 and pep 5) or further purified on a 16.5% SDSpolyacrylamide gel and processed for sequencing as in Matsudaira (1987)(pep 3 and pep 4).

Isolation of HNF-4 cDNA Clones

Oligonucleotide primers corresponding to the least degenerate regions ofpep 1, pep 2 and pep 3 were synthesized: Primer 1S (from sense directionof pep 1) was 5′CC(C/A)tcc(C/G) AXGGNGCNAAYYTNAA-3′ (SEQ ID NO:2) whereN=A+G+T+C, X=A+G, Y=C+T. Primer 1A (antisense of pep 1) was5′-TTAggTTNGCNCCYT(G/C)N(G/C)XNGG-3′(SEQ ID NO:3). Primer 2S (sense ofpep 2) was 5′-CATCTAGAATtGAgCAgAT(Y/A)CA(G/A)TTYAT(Y/A)AA-3 ′ (SEQ IDNO:4). Primer 2A (antisense of pep 2) was 5′AACGTCAGAgcTT(XIT)AT(G/A)AAYTG(XIT)ATYTGYTC-3′ (SEQ ID NO:5). Primer 3S (sense of pep 3) was5′-GAgGCtGTNCAXAAYGAX(C/A)GNGA-3′ (SEQ ID NO:6). Primer 3A (antisense ofpep 3) was 5′-TC(Y/G)C(G/T)cTCXTTYTGNACNGCYTC- (SEQ 1D NO:7). Lower caseletters indicate codon usage according to Lathe (1985); underlinedregions indicate an Xho 1 restriction site used for subcloning. Theprimers were used in the polymerase chain reaction (PCR) (Saiki et al.,1988) in pairwise combinations (Primer 1S+2A, 1S+3A, etc.) following theprotocol by Perkin-Elmer Cetus. 50 μl-reactions containing 0.5 to 4 μgof each primer (1S and 1A, 4 μg; 2S and 2A, 0.5 μg; 3, 1 μg; 3A, 1.5 μg)and 10 μl of rat liver cDNA library in λ Zap II (from Strategene,1.5×10⁶ independent recombinants, amplified and used at 4×10¹⁰ pfu/ml)underwent 30 cycles in a DNA Thermal Cycler (Perkin Elmer Cetus). Eachcycle consisted of 1 mm. at 94° C., 1 mm. at 57° C., 2.5 mm. (plus 5sec/cycle) at 72° C. PCR products were cloned into the polylinker regionof Bluescript KS(+) (Stratagene) and sequenced using the Sequenase kitfrom U.S. Biochemicals. d1TP reactions were performed on regions wherethe sequence was ambiguous.

The nonamplified rat liver cDNA library (Stratogene) was screened forfull length clones as described in Maniatis et al. (1982) except: thenitrocellulose filters were autoclaved to bind the DNA; no formamide wasused in the prehybridization buffer; and hybridization and washings weredone at 50° C. The probe was the subcloned PCR product obtained withPrimers 3S and 2A labeled with ³²P by random priming (Feinberg &Vogelstein, 1983).

Transactivation Assay

The HIV-CAT reporter construct (˜5 kb) contained −57 to +80 of the humanimmunodeficiency virus (HIV) long terminal repeat (LTR) (Rosen et al.,1985) immediately 5′ to the bacterial chloramphenicol acetyl transferase(CAT) gene linked to the SV40 splice and poly(a) sites (from pSV2 CAT,Gorman et al., 1982) in pGEM-1 (Promega) (construction described in Lew,Decker, Stehlow, Darnell, in preparation). The APF1-HIV-CAT reporterconstruct consisted of two APF1 oligonucleotides in direct repeat clonedinto the Sma 1 site of the pGEM polylinker (17 bp form the HIV LTR) ofHIV-CAT. The HNF-4 expression vectors (sense, pLEN4S, and antisense,pLEN4A) were constructed by cloning the entire 3 kb HNF-4 cDNA of pf7into the BamH 1 site of pLEN (courtesy of Cal-Bio Inc.) pLEN is a ˜5 kbexpression vector containing the SV40 enhancer (1120-bp, Hind IIIfragment), the human metallothionein promoter (836-bp, Hind III-BamH1fragment) and human growth hormone 3′ untranslated region (˜550-bp, BamHI-EcoR I (fragment) in pUC8.

DNA transfections and β-galactosidase and CAT assays were performedessentially as in Sambrook et al. (1989). DNA was transfected into HeLacells, grown in Dulbecco's-Modified Eagle's medium (DMEM, Gibco) plus10% bovine calf serum (BCS, Hyclone), using the calcium phosphatemethod. A precipitate of HNF-4 expression vector (pLEN4S or PLEN4A, 0 to5 μg), 1 μg pCMV-β(gal) (internal control, MacGregor & Caskey, 1989), 2μg reporter construct (HIV-CAT or APF1-HIV-CAT) and 50 μg denaturedsonicated salmon sperm DNA were added to cells 60-80% confluent in a100-mm dish. After 15 hrs. at 37° C., the cells were treated with aglycerol shock (15%) and incubated for 48 hours at 37° C. in DMEM plus10% BCS and 10 mM sodium butyrate (to enhance expression from the SV40enhancer, Gorman et al., 1983). Extracts were prepared, normalized toβ-galactosidase activity and assayed for CAT activity (20-hr. incubationat 37° C.).

Northern Blot Analysis

Total RNA was extracted from male rat (Sprague-Dawley) tissue using theacid phenol method of Chomezynski and Sacchi (1987) as modified byPuissant and Houdebine (1990). Poly A+ RNA was selected on oligo-dTcellulose columns and electrophoresed (5 μg/lane) in a 1% agaroseformaldehyde gel as described in Sambrook et al. (1989). The RNA wastransferred to Immobilon-N (Millipore) and probed according to theprotocol provided by the manufacturer. HNF-4 mRNA was detected with arandom-primed cDNA fragment containing nucleotides 616 to 1114 (thehatched area in FIG. 3, top). The high stringency wash was with 0.2×SSC,0.1% SDS at 600° C. for 15 minutes. The autoradiograph with the HNF-4probe was exposed for 3 days with two intensifying screens. RibosomalRNA (28S and 18S, 4.9 and 1.9 kb, respectively) was used as sizemarkers.

Table 1

The sequence and origin of the top strand of the oligonucleotides usedare given. The underlined nucleotides were added for convenience.Complementary bottom strands had four-base overhands at their 5′ ends.The bold type highlights the region of consensus and shows matches inthe hormone response elements. ERE is from the Xenopous vitellogenin A₂(Klein-Hitpaβ et al., 1986), TRE and GRE are palindromic variants of theresponse elements in the rat growth hormone (Glass et al., 1988) andtyrosine aminotransferase (Strahle et al., 1987) genes, respectively.Arrows indicate conserved palindromic regions.

TABLE 1 Oligonucleolides Used in This Study Gene Sequence Position −151to −130 TTR 5′-TCGAGGCAAGGTTCATATTTGTGTAG-3′ −151 to −130 (mouse) (SEQID NO.9) HNF4P TTR 5′-TCGACCCTAGGCAAGGTTCATATGGCC-3′ −156 to −138(mouse) (SEQ ID NO:10) HNF4D TTR 5′-TCGACTCTCTGCAAGGGTCATCAGTAC-3′   −1.86 kB (mouse) (SEQ ID NO:11) APF1 apoCIII5′-TCGAGCGCTGGGCAAAGGTCACCTGC-3′  −66 to −87 (human) (SEQ ID NO:12)LF-A1 a1-AT 5′-AGCAAACAGGGGCTAAGTCCACTGGCTG-3′ −101 to −128 (human) (SEQID NO:13) HNF-4 Consensus GGCAAAGGTCAT(SEQ ID NO:14) T  T G TC C(SEQ IDNO:14) Hormone Response Elements ERE (estrogen)

(SEQ ID NO:15) TRE (thyroid)

(SEQ ID NO:16) GRE (glucocorticoid)

(SEQ ID NO:17)

Results

Purification and Characterization of HNF-4 Protein

Table 1 lists the different oligonucleotides used in the purificationand characterization of the HNF-4 binding protein. Oligonucleotide −151to −130 contains the HNF-4 site (−151 to −140) required for TTRexpression in transfection assays as well as a weak HNF-3 site (−130 to−140) (Costa et al., 1989); HNF4P is similar to −151 to −130 but doesnot contain the HNF-3 site; HNF4D is from a distal site in the TTRpromoter (approximately −1.9 kb) which was shown to enhance thetranscription of TTR marginally (Costa et al., 1988; 1989) and which isbound less well by protein in crude liver extracts than HNF4P. APF1 andLF-A1 are oligonucleotides derived from the promoter regions of thehuman apolipoprotein CIII (apoCIII) and α1-antitrypsin (α1-AT) genes,respectively. Cross competition studies done previously (Costa et al.,1990) showed that the factor that binds to the HNF-4 site in the TTRpromoter also binds to APF1.

HNF-4 binding protein was purified from rat liver nuclear extract by sixchromatography steps including sequence-specific DNA affinity columnsmade with either multimeric HNF4P or APF1 oligonucleotides. Each stepwas assayed by the mobility-shift assay using a double-stranded probe(HNF4P or APF1). An SDS gel of the starting material of the last fivecolumns plus the final purified fraction (Fxn 38, FIG. 1A) showed asingle Coomassie-stained band of 54 kD nominal molecular weight thatco-purified with the mobility-shift activity. In one preparation,approximately 700 mg nuclear protein from 41 rats yielded 30-40 μg ofthe 54 kD protein with an overall recovery of 10-15% based on themobility-shift activity. By comparing protein concentration andDNA-binding activity (APF1 probe) for each step of the purification, thecumulative gain in specific activity was estimated to be 5000 to10,000-fold.

To show that the 54 kD species was the HNF-4 binding protein, thepurified material was subjected to preparative SDS-PAGE, the gel was cutinto slices and the proteins were eluted from each slice, renatured andassayed for HNF-4 binding activity. One such renaturation experiment inwhich only the 45 to 65 kD region was assayed showed that the major bandmigrating at 54 kD (primarily slice 3) contained HNF-4 binding activity(FIG. 1B). Other experiments (not shown) verified that the regions below45 kD and above 65 kD did not contain binding activity.

The affinity column containing the apoCIII site, APF1 (oligo #2, FIG.1A) was used in the purification scheme after the column with the TTRsite, HNF4P (oligo #1, FIG. 1A). Therefore, to be certain that the finalpurified material still bound the TTR site, four different probescontaining slightly different HNF-4 sites (APF-1, −151 to −130, HNF4P,HNF4D) and three probes lacking sequence similarity to the HNF-4recognition site (−175 to −151, HNF3 and C/EBP) were labeled to the samespecific activity and tested in the mobility-shift assay with thepurified protein. The purified material bound to all four HNF-4 sitesand product identical shift bands (FIG. 1B). The different relativeaffinities of the purified material for the various probes (APF1>−151 to−130=HNF4P>HNF4D) is the same as that found in crude liver nuclearextracts (not shown). As expected, the purified material did not bind toany of the unrelated oligonucleotides (FIG. 1B, lanes 9-14).

To verify that the protein we purified was the one originally describedby Costa et al. (1989), the purified protein was shown to protect theregion from −140 to −150 of the coding strand in the TTR promoter fromcleavage by copper phenanthroline (FIG. 2A). This is the same regionoriginally defined as the HNF-4 site by transient transfection assayswith deletion mutants and by methylation interference experiments withcrude liver extracts (Costa et al., 1989).

The appearance of minor bands migrating slightly faster than the majorband at 54 kD in some silver-stained SDS gels (evident as a broad bandin FIG. 1C) and the fact that the purified material bound severalsomewhat different probes raised the concern that there might be morethan one DNA binding protein present in the purified material. Toexamine this possibility, Mono Q fraction 38 was treated with amodifying reagent (phosphatase or one of several proteases), dividedinto aliquots and subjected to the mobility-shift assay using the fourHNF-4 probes described above. The results, displayed in FIG. 2B, showthat a given treatment (calf intestine alkaline phosphatase (CIP),Protease V8 (V8), Endoproteinase Lys-C (lysC)) created essentially thesame pattern of shifted bands regardless of the probe used. Had thepurified material contained a mixture of different polypeptides,different peptide fragments, and therefore different shift bands, shouldhave resulted. Therefore, we concluded that there was a singlepolypeptide in the purified material that bound to the various probes.

Isolation of HNF-4 cDNA Clones

In order to isolate the cDNA encoding HNF-4 protein, a partial aminoacid sequence of the protein purified from the rat liver was obtained.Since the intact protein was found to be N-terminally blocked, thepurified material (Mono, fxn 38; 10 μg) was subjected to reverse-phasehigh pressure liquid chromatography (HPLC) and the major peak,containing the 54 kD protein, was cleaved with cyanogen bromide. Theresulting peptides were separated by HPLC and sequenced.

Five peptide sequences were obtained (pep 1-5). Sense (S) and antisense(A) primers 23 nucleotides long with degeneracies ranging from 36 to4096 were made to three of the peptides (pep 1, pep 2, pep 3). Theprimers were used in pairwise combinations (primers 1S and 2A, 1A and2S, etc.) in a polymerase chain reaction (PCR) with an amplified ratliver cDNA library a the template. Only the combinations of primers 1Sand 2A and primers 3S and 2A resulted in products easily discernible byethidium-bromide staining of an agarose gel (1.0 and 0.5 kilobase, kb,respectively). After subcloning and sequencing, the large product(1S+2A) was found to contain the smaller product (3S+2A) (FIG. 3, top).The deduced amino acid sequence from the large product also contained aregion very similar to the two zinc fingers found in steroid hormonereceptors. The shorter PCR product, which did not contain the zincfingers, was used to screen 3.6×10⁵ primary recombinants in the ratliver library. Of 22 positive clones at the second round of screening,nine were fully characterized and found to be overlapping.

The partial nucleotide sequence of the largest cDNA insert (pf7, FIG. 3bottom) contains a long open reading frame of 1365 base pairs (bp)starting with an initiator methionine at nucleotide 59. There is anotherin-frame ATG codon beginning at nucleotide 32 but comparison with theconsensus sequence for translation initiation (GCC A/G CCATGG, Kozak,1987) and SDS-PAGE analysis of in vitro translation products (not shown)suggest that the ATG codon at nucleotide 59 is the major initiator fortranslation. All five peptide sequences derived from the purified HNF-4protein appeared in the predicted amino acid sequence (FIG. 3 bottom)confirming that the purified HNF-4 preparation did indeed contain onlyone major polypeptide. The 1365-bp open reading frame encodes a protein455 amino acids long with molecular weight of 50.6 kD. Thepolyadenylation signal was not found.

A search of GenBank revealed that HNF-4 is a novel protein but that ithas a structure analogous to that of the steroid/thyroid hormonereceptors (see FIG. 4). HNF-4 contains a region with two potential zincfingers between amino acids 50 and 116 which is 40 to 63% identical tothe zinc finger (DNA binding) domain of other members of the steroidreceptor superfamily. The proposed regulatory protein for the mousemajor histocompatibility class I proteins (H-2RIIBP (Hamada et al.,1989) had the greatest similarity (62.7% identity) and the human thyroidhormone receptor (c-erbA; T₃T_(β)) (Weinberger et al., 1986) was thesecond most similar (59.7% identity) in this region. While the zincfinger domain of HNF-4 is flanked by regions with no similarity to anyknown protein, there is a large hydrophobic region in the C-terminalhalf of the protein (amino acids 133 to 373) which has definitesimilarity to the ligand binding domain of some of the other receptors(20-37% identity). Again, HNF-4 is most similar to H-2RIIBP (37.3%identity) but as with H-2RIIBP, it is not known if HNF-4 requires aligand let alone what the ligand might be.

The HNF-4 protein has two other distinctive features: a proline-richregion (23%) at the C-terminus (amino acids 400-477) which could be anactivator domain (Mermod et al., 1989) and three serine/threonine-richregions (30-38%) scattered throughout the molecule (amino acids 15 to44, 129 to 161, and 398 to 426) which could be sites for phosphorylation(Krebs et al., 1988). Whether or not HNF-4 is modified has not beenestablished yet, but the possibility of some post translationalmodification is suggested by the somewhat aberrant mobility of theprotein isolated from rat liver in the SDS gel (54 kD versus 50.6 kDpredicted from amino acid sequence) as well as the appearance of minorbands migrating slightly faster than the major band in SDS gels.

In vitro Expression of HNF-4 cDNA

To verify that the cDNA clone pf7 encoded the HNF-4 binding protein, T7RNA polymerase transcripts were produced and translated in vitro and theresulting protein was tested in the mobility-shift assay. The proteinsynthesized in vitro bound the APF-1 oligonucleotide in asequence-specific manner (lanes 3 and 4, FIG. 5B) with the shiftedcomplex migrating at a position identical to that of the complex formedwith the material purified from rat liver (compare lane 3 to 1, FIG.5B). The position of the stop codon was confirmed by cutting the pf7cDNA at unique restriction sites either before (PflM 1, nucleotide 1309)or after (Sph I, nucleotide 1584) the proposed stop codon (nucleotide1424) and then synthesizing the protein in vitro and preforming amobility-shift assay. The product of the template cut with Sph Iproduced a complex similar to that produced by the full-length cDNA (XhoI), but the PflM I-cut template yielded a faster moving complex (lanes3, 5, 7; FIG. 5B). Analysis of the protein products on an SDS gel showedthat the product from the Sph I-cut template was the same size as thatfrom the full length template (compare lane 2 to 1, FIG. 5C) and thatboth migrated at a position roughly equivalent to that of the purifiedrat nuclear protein—54 kD. The product of the PflM I-cut templatemigrated faster, confirming the prediction that it should be 36 aminoacids (4000 daltons) shorter (lane 3, FIG. 5C). Plasmid template cutwith Hga I (at nucleotide 1171) produced an even shorter protein product(by 45 amino acids, 5175 daltons) (lane 4, FIG. 5C) which gave rise to afaster migrating shift complex (lane 9, FIG. 5B). When the truncated invitro translation products were tested for DNA binding to anoligonucleotide containing another HNF-4 site, HNF4P, identical resultswere obtained (gel not shown). The results of the in vitro translationexperiments confirm that the pf7 cDNA encodes a protein that binds tothe HNF-4 recognition site in a fashion analogous to that of thepurified protein.

HNF-4 Binds to Its Recognition Site as a Dimer

Further examination of translation products produced from truncated cDNAtemplates showed that a polypeptide containing amino acids 1 to 219 (HphI-cut, lane 5, FIG. 5C) did not bind DNA even though the entire zincfinger region, the DNA binding domain of the receptors, was present(lane II, FIG. 5B). Thus, the region between amino acid 219 and 374, thepossible ligand-binding domain, might be required for binding of theHNF-4 protein to its recognition site. Since amino acids in the ligandbinding domain of the estrogen receptor are known to be necessary forreceptor dimerization and subsequent DNA binding (Kumar & Chambon, 1988;Fawell et al., 1990), we determined whether HNF-4 binds to itsrecognition site as a monomer or as a dimer. The full length cDNA (XhoI) was co-translated in vitro with either of the two truncated productsthat bind DNA (PflM I and Hga I) and the products were tested in themobility-shift assay. When the full length and truncated transcriptswere translated together, complexes of intermediate mobility wereproduced with both the APF-1 probe (lanes 3 and 5, FIG. 5D) and the −151to −130 TTR probe (not shown). These intermediate bands were most likelyproduced by heterodimers between the full length and truncated proteinswhich suggests that the shift complex that was monitored consists of ahomodimeric protein bound to the probe. Since no shift complexescorresponding to monomers were detected with either the in vitrotranslated or the purified protein and since the transcript lacking theproposed domain (Hph I) did not bind the probe at all, we conclude thatprotein dimerization is required for HNF-4 to bind to its recognitionsite.

Transcriptional Activation by Cloned HNF-4

Since deletion of the HNF-4 binding site in the TTR promoter severelyreduced transcription of transfected templates (Costa et al., 1989), wedetermined whether HNF-4 produced from the cloned cDNA would activatetranscription of a target gene. An expression vector containing HNF-4cDNA was cotransfected into HeLa cells with constructs containing areporter gene, chloramphenicol acetyl transferase (CAT), which eitherdid or did not contain HNF-4 recognition sites (APF1-HIV-CAT andHIV-CAT, respectively). The results are shown in FIG. 6. The HNF-4expression vector containing the cDNA in the sense orientationstimulated CAT production from the reporter constructs only when theHNF-4 sites were present (compare lanes 2-4 to lanes 6-8, FIG. 6). Thevector containing the cDNA in the antisense orientation, on the otherhand, did not activate CAT expression above background (compare lanes9-11 to lane 1, FIG. 6). Thus, we concluded that, under the conditionsof these experiments, HNF-4 protein can activate transcription of atarget gene. Furthermore, since the cells in which the activationoccurred were non-hepatic in origin, no liver-specificpost-translational modifications seem to be necessary for HNF-4function.

Tissue Distribution of HNF-4 mRNA is Limited

HNF-4 binding activity was first found in liver. Since then, it has alsobeen found in kidney and intestine but not in spleen or brain (Costa etal., 1990). To see if the tissue distribution of the HNF-4 bindingactivity reflected that of HNF-4 mRNA and to determine the size of theHNF-4 mRNA, a Northern blot analysis was performed. As shown in FIG. 7,the HNF-4 mRNA is present as a single species in rat liver, kidney andintestine but is absent in spleen, brain, white fat, lung and heart.This result supports the conclusion that HNF-4 is neither presentexclusively in liver nor present in all tissues. The size of the mRNAwas the same, −4.5 kB, in all rat tissues as well as in mouse liver(lane 1, FIG. 7). This is consistent with the fact that the pf7 cloneisolated from the rat liver cDNA library contains a cDNA insertapproximately 3 kb long but does not contain a polyadenylation site. Aweak signal at approximately 2.3 kb was also seen (lanes 2, 3—FIG. 7).It varied in amount between blots; its relation to the major signal, ifany, is not known.

HNF-4 Binds to an LF-A1 Site

LF-A1 is a liver-enriched factor that binds to a site required fortranscription of human α1-antitrypsin (Monaci et al., 1988; HNF-2 in Liet al., 1988) certain apolipoproteins and other genes expressed inhepatocytes (Hardon et al., 1988; Vaulont et al., 1989). Since the LF-A1sites are similar in sequence to the HNF-4 binding sites (see Table 1),we used the mobility-shift assay to test the affinity of the HNF-4protein for one of the LF-A1 sites (FIG. 8). HNF-4 protein, eitherpurified from rat liver or translated in vitro from the HNF-4 cDNA,bound the LF-A1 probe very well, producing a shift complexindistinguishable from those formed with the APF1 and HNF4P probes(compare lane 3 and 9 to 1 and 5 and 7 and 11, respectively—FIG. 8). Infact, the LF-A1 probe gave a stronger signal than the HNF4P probe (allprobes were labeled to the same specific activity). To see whether themajor protein species that binds the LF-A1 site in crude extracts is thesame as that which binds the probe to purify HNF-4 protein, themobility-shift assay was carried out with crude rat liver nuclearextracts. The results show that the major shift complex that was formedwith the LF-A1 probe migrated at a position identical to that formedwith the APF1 probe (compare lane 16 to 13, FIG. 8). In addition, theLF-A1 and APF1 complexes were specifically completed by each other(lanes 15 and 18, FIG. 8) and, as with the purified and in vitroproduced HNF-4 protein,t he LF-A1 site appeared to have a somewhat loweraffinity for the factor than the APF1 site. Thus, it appears that HNF-4could be identical to LF-A1.

HNF-4 does not Significantly Bind ERE, TRE or GRE

Since the zinc finger region of HNF-4 is very similar to that of thethyroid and thyroid hormone receptors and since the APF1 site containshalf of the palindrome found in those response elements (AGGTCA), wetested the HNF-4 protein for binding to estrogen, glucocorticoid andthyroid hormone response elements (ERE, GRE, TRE, respectively, seeTable 1) by competition of these sites for labeled APF-1 probe. None ofthe three hormone response elements significantly blocked complexformation with the APF1 probe (gel not shown). since HNF-4 protein has avery high affinity for the APF1 site, we increased the sensitivity ofthe assay by using as a probe an oligonucleotide for which HNF-4 has alower binding affinity, −150 to −130 TTR (see FIG. 1B). The results,shown in FIG. 9, indicate that the GRE and the TRE did not compete thecomplex formation by the −151 to −130 TTR probe significantly betterthan a completely unrelated oligonucleotide (015; lanes 11-18). On theother hand, the ERE did compete slightly better than the unrelatedoligonucleotide (compare lanes 8 and 19 to 17 and 18) but not nearly aswell as the oligonucleotide containing the weakest HNF-4 site known todate (HNF4D) (compare lanes 8-10 to 5-7). Since all these competitionswere in high molar excess (50-, 250- and 500-fold), we conclude thatHNF-4 does not bind either the GRE, TRE or ERE to a degree which wouldbe likely to be relevant in vivo.

Discussion

The invention in its primary aspect comprises the protein purificationof and the cloning and sequencing of a cDNA for a new tissue-restrictedmammalian transcription factor termed hepatocyte nuclear factor 4(HNF-4). HNF-4 was so named because its presence was first detected inliver extracts but not in extracts from several other tissues and itsrecognition site was distinct from that of three previously describedproteins found mainly in the liver (Costa et al., 1989).

HNF-4—A Novel Member of the Steroid Hormone Receptor Superfamily

The deduced amino acid sequence of the HNF-4 protein indicates that itis a member of the steroid/thyroid hormone receptor superfamily, an everincreasing group of ligand-dependent transcription factors which possessa high degree of similarity in their DNA binding (zinc finger) domains.While HNF-4 is similar in sequence to the other factors in thezinc-finger domain, it could be a member of a new subfamily. The membersof the superfamily have been classified according to the amino acidsequence in the knuckle of the first zinc finger (between C₃ and C₄)(referred to as the P Box), a region important in recognizing thesequence of the half site of the palindrome in hormone response elements(Danielson et al., 1989; Mader et al., 1989; Umesono & Evans, 1989;Forman & Samuels, 1990). For example, members of the thyroid hormonereceptor (TR) subfamily contain amino acids EGCKG and bind to a TREwhile members of the estrogen receptor (ER) subfamily contain aminoacids EGCKA and bind to an ERE. The sequence of HNF-4 in this region(DGCKG) is most similar to that of the TR subfamily except that itcontains an aspartic acid (D) in place of a glutamic acid (E) followingC₃. This could explain why HNF-4 does not bind to a TRE (FIG. 9) eventhough it is almost identical ( 9/12 residues) to the HNF-4 consensussite. The significance, if any, of the very marginal binding of HNF-4 tothe ERE (FIG. 9) is not known. While HNF-4 is the only factor publishedto date with the DGCKG sequence, considering the sizes of the othersubfamilies, we anticipate that more will be found in the future (seereceptors compiled in Umesono & Evans, 1989; Forman & Samuels, 1990;hap, de The et al., 1987; H-2RIIBP, Hamada et al., 1989; N10, Ryseck etal., 1989).

Like the well-characterized receptor proteins (estrogen, Kumar &Chambon, 1988; Fawell et al., 1990; thyroid hormone and retinoic acid,Forman et al., 1989; glucocorticoid Tsai et al., 1988), HNF-4 proteinbinds to its recognition site as a homodimer (FIG. 5D), even though thatsite lacks obvious dyad symmetry. Receptor dimerization in the otherreceptors has been localized to a series of heptad repeats ofhydrophobic residues in the ligand-binding domain (Forman et al., 1989;Fawell et al., 1990; Forman & Samuels, 1990). The corresponding regionin HNF-4 is also required for DNA binding (FIG. 5B) and contains atleast twelve heptad repeats. Homodimer formation raises the possibilityof heterodimer formation between HNF-4 and other transcription factors,as has been seen between the thyroid hormone and retionic acid receptors(Forman et al., 1989; Glass et al., 1989).

Since TTR expression is not dependent on hormone regulation, we did notanticipate that HNF-4 would fall into this ligand-dependent superfamily.However, its membership in this family and its limited homology to theligand binding domains of other receptors with known ligands, raises thepossibility that HNF-4 has an as yet unidentified ligand. Consideringthe number and variety of genes that HNF-4 controls (discussed below),the possibility of a ligand for HNF-4 is of considerable interest.Nonetheless, since so many other members of the superfamily fall intothis category of “orphan receptors”—proteins for which no ligand hasbeen identified (e.g., COUP-TF, Wang et al., 1989, ear2, Miyajima etal., 1988; ERR, Giguere et al., 1988; H-2RIIBP, Hamada et al., 1989;N10, Ryseck et al., 1989), one must also entertain the possibility thatthese receptors have no ligands. Since the ligand binding domainoverlaps with the dimerization domain, similarity in this region couldhave been maintained only for the purpose of dimerization and not forthe purpose of binding a ligand.

HNF-4, LF-A1 and AF-1

LF-A1 is a liver-enriched factor originally identified in theα1-antitrypsin gene promoter (Li et al., 1988; Monaci et al., 1988) as asite conferring positive transcription regulation in vivo and in vitro.LF-A1 sites have been found also in the regulatory regions of theapolipoprotein A1 gene, haptoglobin-related genes (Hardon et al., 1988)and the pyruvate kinase L-type gene (Vaulont et al., 1989). In thispaper we present DNA binding data that suggest that HNF-4 could beidentical to LF-A1. However, since there are several examples of morethan one factor binding to a given enhancer element, particularly amongthe hormone receptors (reviewed in Wingender, 1990; Ahe et al., 1985;Mueller et al., 1990; Schule et al., 1990; Umesono et al., 1988),positive identification of HNF-4 as LF-A1 must await furtherpurification of LF-A1.

An example of a factor that appears to be distinct from HNF-4 but whichhas the same binding specificity as HNF-4, is AF-1 (apolipoproteinfactor 1) which regulates the human apoCIII and apoB100 genes (Reue etal., 1988; Leff et al., 1989). While AF-1 purified from mouse liverbinds to the −151 to −130 TTR oligonucleotide and footprints, the sameregion of the apoCIII promoter as does the purified HNF-4 protein, thetissue specificity and chromatographic properties of the two factorsappears to be disparate (T. Leff, F. M. Sladek, unpublishedobservations). Regardless of whether HNF-4 is identical to or distinctfrom LF-A and AF-1, since HNF-4 binds to their recognition sites withrelatively high affinity in vitro, one must consider the possibilitythat HNF-4 might also act on these sites in vivo. HNF-4 could be one ofseveral potentially competing DNA binding proteins that interact with aseries of related sites from a variety of genes transcribed in theliver.

HNF-4 and Liver-specific Gene Expression

A primary objective of the present invention is to identifytranscription factors that are themselves transcriptionally controlledin the liver. HNF-4 appears to be such a factor: HNF-4 can activatetranscription in cells that are not of hepatic origin (FIG. 6)indicating that no liver-specific modifications are required for HNF-4function, and HNF-4 mRNA is absent in many tissues (FIG. 7). Theseresults, taken together with the demonstration that the rate of HNF-4gene transcription is high in the liver but negligible in other tissues(Xanthopoulos, Prezioso, Chen, Sladek, Darnell, in preparation),indicate that HNF-4, like HNF-3 (Lai et al., 1990) and C/EBP(Xanthopoulos et al., 1989), is a transcriptionally controlledtranscription factor. Antecedent regulatory genes in a regulatorycascade can now be sought with confidence by studying the factors thatregulate the genes that encode these regulatory proteins.

The investigation of tissue specific expression has ruled out, to agreater or lesser degree, two simple hypotheses which were entertained.First, there is no universal liver-specific transcription factor orgroup of transcription factors: HNF-1, C/EBP, HNF-3 and HNF-4 all havebinding sites on several genes but none is a “master” positive-actingfactor. Indeed, all of these factors are present in tissues other thanliver and some are even in tissues not of the same germline as the liver(HNF-1, also in kidney and spleen, Baumeueter et al., 1990; C/EB, brain,fat, intestine, lung and skin, Birkenmeier et al., 1989; Xanthopoulos etal., 1989; Kuo et al., 1990; Ruppert et al., 1990; HNF-3A, intestine insmall amounts; HNF-4, kidney and intestine, FIG. 7). In addition tovarying in their tissue distribution, these factors have proteinstructures that classify them as members of four distinct groups ofregulators, none of which is found exclusively in the liver (HNF-1,homeo domain; C/EBP, leucine zipper; HNF-3, unclassified; HNF-4, steroidhormone receptors). Second, we cannot immediately understand the logicthat unites the group of genes that a particular factor may helpregulate. For example, HNF-4 apparently acts positively on genesencoding apolipoproteins, which are involved in cholesterol homeostasis,transthyretin, which carries thyroid hormone and Vitamin A in the serum,as well as α1-antitrypsin, a protease inhibitor, pyruvate kinase, whichplays a role in glycolysis, and glutamine synthetase, which acts inamino acid biosynthesis (C. F. Kuo, F. M. Sladek, unpublishedobservations). Why this factor is involved in regulating this variedassortment of genes is far from obvious.

The invention has been described in detail, setting forth the preferredembodiments. However, alternative embodiments are contemplated asfalling within the invention. Consequently, the scope of the claims isnot to be limited by the teachings contained herein.

1. A competitive assay for binding activity to recombinant HNF-4receptors, comprising: (a) reacting a suspected ligand with a fragmentof a recombinant HNF-4 receptor that retains ligand binding activity,wherein the fragment of the recombinant HNF-4 receptor that retainsligand binding activity consists of twelve heptad repeats of SEQ ID NO:18 or amino acids 133-373 of SEQ ID NO: 18, in the presence of a knownquantity of a labeled competing ligand; and (b) comparing the amount ofbound label in the presence of the suspected ligand to that observed inits absence.
 2. The assay of claim 1, wherein the labeled competingligand is labeled with a radioimmunoconjugate.
 3. The assay of claim 1,wherein the labeled competing ligand is labeled by fluorescence.